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Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
439
Single-domain antibody fragments derived from heavy-chain antibodies: a review
L. Eyer, K. Hruska
Veterinary Research Institute, Brno, Czech Republic
ABSTRACT: Single-domain antibody (sdAb) fragments derived from heavy-chain antibodies of camelids and cartilagi-nous fish represent a new generation of therapeutic agents and immunoreagents. Due to their unique characteristics, such as low molecular weight, high physical-chemical stability, good water solubility, and the ability to bind antigens inaccessible to conventional antibodies, they could potentially act as a substitute for conventional therapeutic drugs in the treatment of serious human diseases, and, moreover, could be broadly used in analyses and diagnostics. In this review article, an analysis of 826 publications oriented to heavy-chain antibodies and their sdAb fragments indexed in the Web of Science® database since 1993 has been carried out. Attention has predominantly been paid to papers published from 2010 to June 2012. Key publications are presented in tables and are characterised by descriptive words, abstracts and references. The presented publications have been sorted according to seven basic criteria: review articles and monographs, heavy-chain antibodies of camelids and sharks, production of sdAb fragments using recombinant technology, characteristic properties of sdAb fragments, application of sdAb fragments in therapy, application of sdAb fragments in diagnostic and immunoanalytical methods and other prospective uses of sdAb fragments. This review article should highlight the typical properties of heavy-chain antibodies and sdAb fragments which differentiate them from conventional antibodies and other available recombinant fragments, and also emphasize their extremely broad application potential, mainly in human disease therapy. At the same time it allows an easy and rapid orientation in numerous publications written on this subject, and facilitates the search for the required data.
Keywords: single-domain antibody fragment; heavy-chain antibody; antigen-binding site; camelid; shark; therapy; recombinant technology
Supported by the Ministry of Education, Youth and Sports, Czech Republic (AdmireVet; Grant No. CZ 1.05/2.1.00/01.0006-ED 0006/01/01) and the Ministry of Agriculture of the Czech Republic (Grant No. MZE 0002716202).
Contents
1. Introduction2. Review
2.1. Methods of searching and publication analysis2.2. Review articles and monographs2.3. Heavy-chain antibodies of camelids and sharks2.4. Production of sdAb fragments using recom-
binant technology2.5. Characteristic properties of sdAb fragments2.6. Application of sdAb fragments in therapy2.7. Application of sdAb fragments in diagnostic
and immunoanalytical methods2.8. Other prospective uses of sdAb fragments
3. References
Tables and figures
Table 1. Authors, countries, institutions and jour-nals (Top 5) showing the highest publication activities concerning heavy-chain antibodies and single-domain antibody fragments (Web of Science®, 826 papers from 1993 to June 2012)
Table 2. Important review articles and monographs concerning heavy-chain antibodies and single-domain antibody fragments
Table 3. Camelid and shark heavy-chain antibodiesTable 4. Production of single-domain antibody
fragments using recombinant technologyTable 5. Characteristic properties of single-domain antibody fragments
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
440
1. Introduction
A surprising discovery was made in the 1990s: in addition to conventional antibodies, mammals of Camelidae family and also some cartilaginous fish have evolved a distinctive type of antibody molecule composed entirely of two identical heavy chains (Hamerscasterman et al. 1993; Greenberg et al. 1995). Because these antibodies are devoid of light chains, they are called heavy-chain antibodies. The antigen-binding site of heavy-chain antibodies is confined to one single domain referred to as the VHH (Variable domain of the Heavy chain of the Heavy-chain antibody) in Camelidae and VNAR (Variable domain of the shark New Antigen Receptor) in car-tilaginous fish. These variable domains can be eas-ily expressed in bacteria, yeasts or in other hosts as recombinant single-domain antibodies (sdAb), which are the smallest available intact antigen-bind-ing fragments. SdAb fragments derived from llama and camel heavy-chain antibodies are often referred to as nanoantibodies or nanobodies®. Due to their biophysical and pharmaceutical properties, which are conferred by their single-domain nature and small molecule size, sdAb fragments offer a broad application potential, especially as new immuno-therapeutic drugs and also as efficient reagents in immunoanalytical and diagnostic methods. The aim of this review is to highlight the unique features of heavy-chain antibodies and sdAb fragments and
show their numerous novel feasible applications in research and in clinical development. Publications, which contain key information concerning heavy-chain antibodies and their sdAb fragments, are in this new designed review presented in tables and are characterized with a few descriptive words, full or shortened abstracts and source references. The text in the tables contains several format imperfec-tions, which exist in the Web of Science® database and are caused by transmission and copying of data between various information sources. The missing format of lower and upper indexes (e.g. Ig(2) instead of Ig2 or 10(–6) instead of 10–6) can be given as a typical example. Special characters, such as α, Å or °C were transcribed as alpha, angstrom or degree C, respectively. The attention is predominantly focused on papers published in the last three years, although some older key papers had been also included. The presented publications have been classified ac-cording to seven basic criteria: review articles and monographs, heavy-chain antibodies of camelids and sharks, production of sdAb fragments using recombinant technology, characteristic properties of sdAb fragments, application of sdAb fragments in therapy, application of sdAb fragments in diagnos-tic and immunoanalytical methods and other pro-spective uses of sdAb fragments. This review article should facilitate orientation in the above mentioned subjects and enable rapid and easy searching for particular data and information.
Table 6A. Application of single-domain antibody fragments in therapy: inhibition of enzymes, toxins and other soluble proteins
Table 6B. Application of single-domain antibody fragments in therapy: activity modulation of cell surface proteins
Table 6C. Application of single-domain antibody fragments in therapy: pathogen neutralisation
Table 6D. Application of single-domain antibody fragments in therapy: intracellular expression of single-domain antibody fragments
Table 6E. Application of single-domain antibody fragments in therapy: oral administration of single-domain antibody fragments
Table 6F. Application of single-domain antibody fragments in therapy: prevention of amyloid plaque formation and protein aggregation
Table 6G. Application of single-domain antibody fragments in therapy: multispecific and multi-functional constructs
Table 6H. Application of single-domain antibody fragments in therapy: humanised single-domain antibody fragments
Table 7. Application of single-domain antibody frag-ments in diagnostic and immunoanalytic methods
Table 8. Other prospective uses of single-domain antibody fragments
Figure 1. The number of publications on heavy-chain antibodies and single-domain antibody frag-ments during the period from 1993 to June 2012
Figure 2. Structure of antibodies. (a) Molecule of con-ventional antibody (down), recombinant fragments (up); (b) molecule of camelid heavy-chain antibody (down), single-domain antibody fragment (up); (c) molecule of shark heavy-chain antibody (down), single-domain antibody fragment (up)
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
441
2. Review
2.1. Methods of searching and publication analysis
The publications were retrieved from the Web of Science® database using the following search profile: Topic = (nanobod* OR nanoantibod* OR “single-domain antibod*” OR “heavy-chain anti-bod*” OR “heavy chain-only antibod*” OR VHH OR IgNAR OR “llama* antibod*” OR “camel* an-tibod*” OR “new antigen receptor”), Timespan = all years or 2010–2012, Citation databases = SCI-EXPANDED, SSCI, A&HCI, CPCI-S, CPCI-SSH, BKCI-S and BKCI-SSH, Chemical Databases = CCR-EXPANDED and IC.
Using the above mentioned search profile, a total of 826 publications were retrieved. The first paper describing the occurrence of heavy-chain antibod-ies was published in 1993 by a Belgium research group. Since 2003, publication activity on the topic has sharply increased (Figure 1). In the last three years, 297 papers were published. In 2012, sixty-four papers have already been published at the time of our manuscript preparation. The older publications are predominantly oriented to the elucidation of the structure and characteristics of heavy-chain anti-bodies and their antigen-binding sites. The latest papers describe the possibilities of practical use of sdAb fragments, especially in medicine and therapy.
The Web of Science® utilities have been em-ployed for search results analysis. Of all the pub-lished papers, original research articles prevail
(78.2%). Our analysis shows that 262 institutions from 47 countries are concerned with the subject of sdAb fragments derived from heavy-chain antibod-ies. Authors, institutions, countries and scientific journals which show the highest publication activi-ties (Top 5) are listed in Table 1.
2.2. Review articles and monographs
Eighteen review articles are listed in Table 2 and characterized by key words (left column), abstracts (in the middle), and references (right column). These papers offer an overall review regarding all important topics, especially about the structure, properties, generation and evolution of heavy-chain antibodies in camelids and sharks (Conrath et al. 2003; Tillib 2011), engineering of sdAb fragments (Deffar et al. 2009), their production, structure and properties (Muyldermans et al. 2001; Harmsen and de Haard 2007), use of sdAb fragments as building blocks for the construction of multivalent and mul-tispecific conjugates (Saerens et al. 2008), applica-tion of sdAb fragments in therapy (Van Bockstaele et al. 2009; Wesolowski et al. 2009) and in im-munoanalytical and diagnostic methods (Huang et al. 2010; de Marco 2011). All these topics are discussed in detail also in original papers, which are presented in Tables 3 to 8. Recently, the new monograph “Single Domain Antibodies” (Saerens and Muyldermans 2012) has been launched with a complete methodology and key protocols for the construction of sdAb libraries and for the selec-
0
20
40
60
80
100
120
140
160
1993 1995 1997 1999 2001 2003 2005 2007 2009 2011
Figure 1. The number of publications on heavy-chain antibodies and single-domain antibody fragments during the period from 1993 to June 2012
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
442
tion and expression of sdAb fragments and their advanced derivatives. This publication hihlights the broad application potential of sdAb fragments and can be used as a practical guide to sdAb recombi-nant technologies.
2.3. Heavy-chain antibodies of camelids and sharks
Heavy-chain antibodies are homodimers of di-sulphide-linked heavy chains (Figure 2) that occur naturally in the serum of camelids and cartilaginous fish. The total amount of heavy-chain antibodies of all serum immunoglobulins has been determined to be about 45% in llama species, while in camels it is about 75% (Hamerscasterman et al. 1993). The function of heavy-chain antibodies in the immune system has not yet been completely explained. Due to the lack of light chains, the antigen-binding site of heavy-chain antibodies is formed by only three complementary determining regions (CDRs), com-pared to six CDRs in conventional antibodies. The CDR3 region of the heavy-chain antibody, which usually plays a crucial role in an antibody-antigen interaction, can be formed by a long finger-like polypeptide loop. Therefore, the antigen-binding site of the heavy-chain antibodies often exhibits a convex shape and differs sharply from the con-cave, groove-shaped antigen-binding site of con-ventional antibodies. With regard to the shape of the antigen-binding site, heavy-chain antibodies recognise preferably epitopes buried in clefts on protein surfaces, e.g. in enzyme active sites, which are usually less antigenic to conventional antibod-ies (Lauwereys et al. 1998; Decanniere et al. 1999; De Genst et al. 2006). The convex shaped antigen-binding site of heavy-chain antibodies is not very suitable for the binding of small molecules (hap-tens). Therefore, only a few papers focused on anti-hapten heavy-chain antibodies have been published to this date (Spinelli et al. 2001; Ladenson et al. 2006; Alvarez-Rueda et al. 2007). Other structural features of heavy-chain antibodies, which differen-tiate them from conventional antibodies, include a higher proportion of hydrophilic amino acids in variable domains (VHH/VNAR), the presence of disulfide bonds stabilizing the antigen-binding site, and an unusually high number of mutational hot spots responsible for structural variability of heavy-chain antibodies (Harmsen and de Haard 2007). In addition to structural studies, recent research
has been focused also on the evolution of heavy-chain antibodies (Nguyen et al. 2002; Flajnik et al. 2011), their possible function in the immune system (Ferrari et al. 2007; Saccodossi et al. 2012), and molecular mechanisms of germline gene segment rearrangement (Nguyen et al. 2000). Some investi-gators have also used heavy-chain antibodies as a suitable alternative to conventional polyclonal an-tibodies, mainly for diagnostic purposes (Anderson and Goldman 2008; Torigoe et al. 2012). Twenty-three papers (eleven of them published from 2010 to 2012) are presented in Table 3.
2.4. Production of sdAb fragments using recombinant technology
Due to their simple modular structure and a single-gene nature, sdAb fragments are easily produced in vitro as recombinant proteins. The production of sdAb fragments is based on the cloning of VHH or VNAR gene segments into phage display vectors, construction of large phage libraries and selection of high-affinity binders us-ing a biopanning process. SdAb fragments are ef-fectively expressed in different microbial hosts, of which Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris are the most commonly used (Makvandi-Nejad et al. 2011; Ezzine et al. 2012; Gorlani et al. 2012). Bacterial species enable high-yield expression of sdAb fragments in the cytosol or in periplasm, whereas yeast cells utilize secre-tory pathways resulting in efficient disulfide bond formation, N-glycosylation and secretion of the recombinant product into the growth medium. Recent papers also describe several alternative strategies for sdAb fragment engineering and pro-duction, such as yeast or ribosome display systems (Yau et al. 2003; Ryckaert et al. 2010), expression of recombinant fragments in filamentous fungi (Joosten et al. 2005), in mammalian cells (Bazl et al. 2007) and in transgenic mice (Zou et al. 2005). Mutagenesis and recombination of CDR regions to improve the affinity and specificity of sdAb fragments have also been discussed (Swain et al. 2010; Fanning and Horn 2011). Current studies are focused on the development of new cloning and expression strategies to produce humanised fragments, multivalent constructs and fusions of antibody fragments to other proteins (Saerens et al. 2005; Vincke et al. 2009; Bell et al. 2010; Pollithy et al. 2011). Selected articles are presented in Table 4.
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2.5. Characteristic properties of sdAb fragments
Recombinant sdAb fragments are formed from only one single polypeptide chain containing about 120 amino acids. Due to their size (4 × 2.5 nm) and molecular weight (12 kDa to 15 kDa), sdAb fragments are the smallest available recombinant antibodies. Their small molecular size enables sdAb to penetrate easily into tissues and inter-cellular spaces, but is, however, the cause of their rapid clearance from the blood by renal filtration (Cortez-Retamozo et al. 2002). The rapid clear-ance of sdAb fragments from the blood by kid-
neys can negatively affect their therapeutic activity. Therefore, several strategies to prolong the half-life of sdAb fragments in serum have been described, e.g. conjugation of sdAb to another recombinant fragment binding specifically to serum albumins or immunoglobulins (Harmsen et al. 2005). SdAb frag-ments that bind to and are internalised by cerebro-microvascular endothelial cells are able to cross the blood-brain barrier and transmigrate into tissues of the central nerve system (Abulrob et al. 2005). Due to their simple monomeric structure, sdAb fragments are conformationally very stable (Dumoulin et al. 2002) and refold easily after heating to achieve their original native structure (Dolk et al. 2005b; Walper et
Figure 2. Structure of antibodies. (a) Molecule of conventional antibody (down), recombinant fragments (up); (b) molecule of camelid heavy-chain antibody (down), single-domain antibody fragment (up); (c) molecule of shark heavy-chain antibody (down), single-domain antibody fragment (up)
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
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al. 2012). They show an increased resistance to high pressure and to low pH (Dumoulin et al. 2002) and maintain their biological activity even in an environ-ment with a high concentration of proteases and detergents (Dolk et al. 2005a; Hussack et al. 2011a). Owing to a higher content of hydrophilic amino acids, sdAb fragments are characterised by good solubility in water and limited agglutination ability (Conrath et al. 2005). Another characteristic feature particularly concerns the affinity of sdAb fragments, which remains usually equivalent to that of the native heavy-chain antibodies. This is a substantial differ-ence from other recombinant fragments, that show often lower affinity compared with their polyclonal or monoclonal counterparts. Eight papers published from 2010 to 2012 describing typical properties of sdAb fragments are presented in Table 5. Eight older articles should also be considered.
2.6. Application of sdAb fragments in therapy
The unique physicochemical and pharmacological properties of camelid and shark sdAb fragments give them a prospective use as new-generation thera-peutic agents for the treatment of serious human diseases. At present, some sdAb-based drugs are in the stage of preclinical testing on animal models or in vitro. Some other drugs have advanced to phase I or II of clinical testing in volunteers. The therapeutic effect of sdAb fragments is particularly based on the following principles: Firstly, a remarkable prefer-ence of sdAb fragments for binding into clefts and cavities on protein surfaces offers the possibility to develop selective therapeutics for efficient inhibition of enzymes (Paalanen et al. 2011), neutralization of proteolytic toxins (Hussack et al. 2011b) and activity modulation of cell surface proteins, such as recep-tors, ion channels and leukocyte ecto-enzymes (Wei et al. 2011; Altintas et al. 2012) involved in cancer and inflammatory diseases. Moreover, sdAb frag-ments recognize also cryptic epitopes hidden deeply in the clefts of virus capsids and in surface envelopes of parasites (Stijlemans et al. 2004; Henderson et al. 2007; Forsman et al. 2008). Secondly, intracellular expression of sdAb fragments as “intrabodies” is a potential strategy to target intracellular antigens, e.g. anti-apoptotic proteins, oncogenes or several viral proteins (Vercruysse et al. 2010). Thirdly, the high structural stability of sdAb fragments in harsh conditions makes them ideally suited for the immu-
notherapy of gastrointestinal disorders using oral ad-ministration (Harmsen et al. 2006; Vandenbroucke et al. 2010). Fourthly, the ability of sdAb fragments to transmigrate across the blood-brain barrier and to prevent amyloid plaque formation could be utilized in the diagnostics and therapy of neurodegenerative diseases (De Genst et al. 2010; Rutgers et al. 2011). Fifthly, a modular nature of sdAb fragments allows an easy engineering of multivalent or multispecific formats that show an increased therapeutic potency compared with monovalent sdAb fragments. Recent research is focused also on the generation of bifunc-tional constructs by coupling of sdAb fragments with enzymes and toxic substances for specific drug de-livery to bacterial or tumour cells (Zhang et al. 2004; Stone et al. 2007). Finally, the non-human origin of camelid and shark sdAb fragments could elicit their neutralisation by the human immune response and a decrease in their therapeutic effect. Therefore, a general strategy to produce humanised sdAb frag-ments has been described as a promising way for lowering the potential risk of immunogenicity of therapeutic sdAb fragments (Vincke et al. 2009). More than half of the 826 analysed papers discuss possible therapeutic applications of sdAb fragments. Key publications are presented in Tables 6A to H.
2.7. Application of sdAb fragments in diagnostic and immunoanalytical methods
Their effective penetration into tissues makes sdAb fragments good candidates for the construction of imaging probes used for in vivo monitoring of tu-mours, metastatic lesions, amyloid fibrils, etc. Such immuno-imaging probes are prepared by the labelling of sdAb fragments with short-lived isotopes, mainly with 99mTc. In contrast to radiolabelled monoclonal antibodies, small sdAb fragments show rapid antigen targeting and fast clearance from blood resulting in a contrast-enhanced imaging signal, reduced accumula-tion of labelled fragments in liver and lower radiation burden. The main limitation of sdAb-based imaging probes, however, is their high non-specific uptake in kidney and bladder (Vaneycken et al. 2010, 2011b). An innovative approach based on sdAb fragments fused to fluorescent proteins and expressed in living cells as “chromobodies” offers the possibility to trace intra-cellular antigens and to modulate protein function in living cells (Schmidthals et al. 2010). Nanoantibodies can also be used in immunoassays and in biosensors
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
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for the quantitative analysis of contaminants and tox-ins in food and environmental samples (Conway et al. 2010; Dona et al. 2010; Kim et al. 2012) and also for diagnostics of serious human diseases (De Marni et al. 2012). Their small size and lower aggregation propensity enable immobilisation of extremely high numbers of nanoantibody molecules on the surface of the well of microtitre plate or biosensor transducer, which leads to a substantial increase in the sensitivity of the immunodetection system. Moreover, highly stable sdAb fragments are resistant to denaturing con-ditions used during biochip regeneration. For details of new publications see Table 7.
2.8. Other prospective uses of sdAb fragments
Besides possible therapeutic, diagnostic and immunoanalytic applications, sdAb fragments
derived from camelid and shark heavy-chain an-tibodies can also be employed as crystallography chaperones to stabilise the conformation of pro-teins during crystallisation trials (Abskharon et al. 2011). Immunoaffinity chromatography represents another prospective field for sdAb fragment ap-plication. SdAb fragments are highly resistant to the effects of organic solvents used for the elution of the captured ligands and for regeneration of im-munoaffinity columns (Franco et al. 2010). Recent applications, such as the development of transport systems across epithelia (Iqbal et al. 2011), inacti-vation of phages infecting dairy bacterial cultures (Hultberg et al. 2007) and mimotope selection strat-egies (Simmons et al. 2008) show the extremely broad application potential of sdAb fragments. Ten recent publications discussing some alternative ap-plications of sdAb fragments are listed in Table 8. This table also includes two older papers which make the review complete.
Table 1. Authors, countries, institutions and journals (Top 5) showing the highest publication activities con-cerning heavy-chain antibodies and single-domain antibody fragments (Web of Science®, 826 papers from 1993 to June 2012)
Item Number of publicationsAuthors (685 in total)Muyldermans S 96Wyns L 32Rahbarizadeh F 28Conrath K 25Tanha J 23Verrips CT 23Institutions (262 in total)Free University of Brussels/Flanders Institute for Biotechnology 188Utrecht University 61National Research Council Canada 47Ablynx NV 32Ghent University 23Countries (47 in total)USA 200Belgium 193Netherlands 115 England 78 France 63Journals (140 in total)Journal of Biological Chemistry 30Journal of Molecular Biology 25Molecular Immunology 24Journal of Immunological Methods 22PLoS ONE 19
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Tabl
e 2.
Impo
rtan
t rev
iew
art
icle
s an
d m
onog
raph
s co
ncer
ning
hea
vy-c
hain
ant
ibod
ies
and
sing
le-d
omai
n an
tibo
dy fr
agm
ents
Cam
elid
imm
unog
lobu
linIg
G c
once
ntra
tion
Sing
le r
adia
l im
mun
odif
-fu
sion
Failu
re o
f pas
sive
tran
sfer
Cam
elid
imm
unog
lobu
lins d
iffer
from
all
othe
r kno
wn
antib
odie
s and
con
trad
ict a
ll co
mm
on th
eori
es o
n an
tibod
y di
vers
ity. I
t was
de
mon
stra
ted
that
up
to 7
5% o
f all
seru
m p
rote
ins a
re im
mun
oglo
bulin
G (I
gG) m
olec
ules
lack
ing
light
cha
ins.
IgG
(2) a
nd Ig
G(3
), w
hich
onl
y co
nsis
t of h
eavy
cha
ins,
hav
e a
low
mol
ecul
ar w
eigh
t whi
ch im
prov
es th
eir b
iodi
stri
butio
n an
d al
low
s a b
ette
r tis
sue
pene
trat
ion.
Of s
peci
al im
port
ance
is th
e lo
ng c
ompl
emen
tary
det
erm
inin
g re
gion
(CD
R) lo
op w
hich
inse
rts d
eep
into
the
activ
e si
te o
f an
enzy
me.
This
bin
ding
pro
pert
y w
as o
nly
obse
rved
in e
xper
imen
ts to
gai
n st
ruct
ural
dat
a an
d to
poi
nt o
ut th
e ex
trao
r-di
nary
val
ue o
f hea
vy c
hain
ant
ibod
ies a
s bio
chem
ical
and
pha
rmac
olog
ical
tool
s. Th
e ac
quis
ition
and
abs
orpt
ion
of a
dequ
ate
amou
nts o
f col
ostr
al im
mun
oglo
bulin
s are
ess
entia
l to
the
heal
th o
f the
neo
nate
, Pre
-col
ostr
um se
rum
IgG
leve
ls in
cam
elid
s are
lo
w, w
ith c
once
ntra
tions
of 0
.26
± 0.
23 m
g/m
l. M
axim
um Ig
G le
vels
are
reac
hed
afte
r 24
h an
d ke
pt a
t a p
late
au w
ith c
once
ntra
-tio
ns o
f 24.
52 ±
8.8
mg/
dl. I
gG c
once
ntra
tions
abo
ve 1
0 m
g/m
l. in
dica
te a
succ
essf
ul p
assi
ve tr
ansf
er. I
gG le
vels
dec
line
afte
r 2–5
w
eeks
and
a m
arke
d in
crea
se is
obs
erve
d be
twee
n on
e an
d tw
o m
onth
s, in
dica
ting
that
the
imm
une
syst
em o
f the
neo
nate
has
st
arte
d to
mat
ure.
A n
umbe
r of d
iffer
ent t
ests
are
ava
ilabl
e fo
r the
ass
essm
ent o
f IgG
seru
m le
vels
. Sin
gle
radi
al im
mun
odiff
usio
n (S
RID
) is t
he o
nly
met
hod
that
spec
ifica
lly m
easu
res s
erum
IgG
con
cent
ratio
ns. I
t is a
relia
ble
assa
y to
test
failu
re o
f pas
sive
tran
s-fe
r (FP
T).
FPT
is a
maj
or fa
ctor
in n
eona
tal m
orta
lity
in c
amel
ids,
but
ver
y lit
tle h
as b
een
publ
ishe
d so
far.
Ther
apeu
tic a
dmin
is-
trat
ion
of c
olos
trum
will
pro
vide
pas
sive
pro
tect
ion
agai
nst i
nfec
tious
dis
ease
s for
a 2
–3-w
eek
peri
od o
f ris
k, a
nd th
e in
trav
enou
s ad
min
istr
atio
n of
20–
40 m
l of c
amel
id p
lasm
a he
lps t
o co
mba
t FPT
.
Wer
nery
20
01
Evol
utio
nJa
wed
-ver
tebr
ates
Shar
ksIm
mun
oglo
bulin
gen
es
Ant
ibod
ies o
f jaw
ed-v
erte
brat
es a
re c
ompo
sed
of p
aire
d he
avy
(H) a
nd li
ght (
L) p
olyp
eptid
e ch
ains
. Sur
pris
ingl
y, th
e se
ra o
f cam
elid
s, nu
rse
shar
k an
d w
obbe
gong
shar
k, a
nd p
ossi
bly
ratfi
sh c
onta
in a
ntib
odie
s tha
t lac
k L-
chai
ns. I
n ca
mel
ids,
thes
e H
eavy
-cha
in a
nti-
bodi
es (H
CA
bs) a
re g
amm
a-is
otyp
es, a
nd a
re fu
nctio
nal i
n an
tigen
bin
ding
. In
this
revi
ew w
e fo
cus o
n th
e de
dica
ted
imm
unog
lobu
-lin
(Ig)
gen
es th
at e
ncod
e th
e H
CA
b in
Cam
elid
ae (c
amel
s, dr
omed
arie
s and
llam
as),
abou
t the
ir or
igin
, and
how
thes
e ca
mel
imm
u-m
oglo
bulin
s evo
lved
and
acq
uire
a la
rge
and
dive
rse
repe
rtoi
re o
f ant
igen
bin
ding
site
s in
abse
nce
of th
e H
-L c
ombi
nato
rial
div
ersi
ty.
Con
rath
et
al. 2
003
Hea
vy-c
hain
ant
ibod
yN
anob
ody
It is
wel
l est
ablis
hed
that
all
cam
elid
s hav
e un
ique
ant
ibod
ies c
ircu
latin
g in
thei
r blo
od. U
nlik
e an
tibod
ies f
rom
oth
er sp
ecie
s, th
ese
spec
ial a
ntib
odie
s are
dev
oid
of li
ght c
hain
s and
are
com
pose
d of
a h
eavy
-cha
in h
omod
imer
.Thes
e so
-cal
led
heav
y-ch
ain
antib
odie
s (H
CA
bs) a
re e
xpre
ssed
aft
er a
V-D
-J re
arra
ngem
ent a
nd re
quir
e de
dica
ted
cons
tant
gam
ma-
gene
s. A
n im
mun
e re
spon
se is
rais
ed in
thes
e so
-cal
led
heav
y-ch
ain
antib
odie
s fol
low
ing
clas
sica
l im
mun
izat
ion
prot
ocol
s. Th
ese
HC
Abs
are
eas
ily
puri
fied
from
seru
m, a
nd th
e an
tigen
-bin
ding
frag
men
t int
erac
ts w
ith p
arts
of t
he ta
rget
that
are
less
ant
igen
ic to
con
vent
iona
l an
tibod
ies.
Sin
ce th
e an
tigen
-bin
ding
site
of t
he d
rom
edar
y H
CA
b is
com
pris
ed in
one
sing
le d
omai
n re
ferr
ed to
as v
aria
ble
dom
ain
of h
eavy
cha
in o
f HC
Ab
(VH
H) o
r nan
obod
y (N
b), w
e de
sign
ed a
stra
tegy
to c
lone
the
Nb
repe
rtoi
re o
f an
imm
uniz
ed
drom
edar
y an
d to
sele
ct th
e N
bs w
ith sp
ecifi
city
for o
ur ta
rget
ant
igen
s. Th
e m
onoc
lona
l Nbs
are
wel
l pro
duce
d in
bac
teri
a, a
re
very
stab
le a
nd h
ighl
y so
lubl
e, a
nd b
ind
thei
r cog
nate
ant
igen
with
hig
h affi
nity
and
spec
ifici
ty. W
e ha
ve su
cces
sful
ly d
evel
oped
re
com
bina
nt N
bs fo
r res
earc
h pu
rpos
es, a
s pro
be in
bio
sens
ors,
to d
iagn
ose
infe
ctio
ns, a
nd to
trea
t dis
ease
s lik
e ca
ncer
or t
rypa
-no
som
osis
.
Muy
lder
-m
ans e
t al.
2009
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
447
Nan
obod
y te
chno
logy
This
shor
t rev
iew
pro
vide
s an
intr
oduc
tion
to th
e ra
pidl
y de
velo
ping
fiel
d of
gen
erat
ion
and
utili
zatio
n of
“cam
el n
anoa
ntib
odie
s”
(or “
nano
bodi
es”)
. The
term
“nan
oant
ibod
y”or
“nan
obod
y” w
as g
iven
to si
ngle
-dom
ain
vari
able
frag
men
ts o
f spe
cial
type
of a
nti-
bodi
es th
at n
atur
ally
exi
st (i
n ad
ditio
n to
cla
ssic
al ty
pes o
f ant
ibod
ies)
in b
lood
of C
amel
idae
fam
ily a
nim
als a
nd in
som
e ch
on-
dric
hthy
an fi
shes
. The
exis
tenc
e of
ver
y effi
cien
t tec
hnol
ogy
of n
anob
ody
gene
ratio
n an
d so
me
very
use
ful c
hara
cter
istic
feat
ures
pr
omis
e a
big
pote
ntia
l for
thei
r use
in im
mun
obio
tech
nolo
gy a
nd m
edic
ine.
Tilli
b 20
11
Nan
obod
ies
Prop
etie
sPr
oduc
tion
App
licat
ions
Nan
obod
ies a
re a
ntib
ody-
deriv
ed th
erap
eutic
pro
tein
s tha
t con
tain
the
uniq
ue st
ruct
ural
and
func
tiona
l pro
pert
ies o
f nat
ural
ly o
ccur
-rin
g he
avy-
chai
n an
tibod
ies.
The
Nan
obod
y te
chno
logy
was
orig
inal
ly d
evel
oped
follo
win
g th
e di
scov
ery
that
cam
elid
ae (c
amel
s and
lla
mas
) pos
sess
fully
func
tiona
l ant
ibod
ies t
hat l
ack
light
cha
ins.
Thes
e he
avy-
chai
n an
tibod
ies c
onta
in a
sing
le v
aria
ble
dom
ain
(VH
H)
and
two
cons
tant
dom
ains
(CH
(2) a
nd C
H(3
)). Im
port
antly
, the
clo
ned
and
isola
ted
VH
H d
omai
n is
a pe
rfec
tly st
able
pol
ypep
tide
har-
borin
g th
e fu
ll an
tigen
-bin
ding
cap
acity
of t
he o
rigin
al h
eavy
-cha
in a
ntib
ody.
Thes
e ne
wly
disc
over
ed V
HH
dom
ains
with
thei
r uni
que
stru
ctur
al a
nd fu
nctio
nal p
rope
rtie
s for
m th
e ba
sis o
f a n
ew g
ener
atio
n of
ther
apeu
tic a
ntib
odie
s whi
ch w
ere
nam
ed N
anob
odie
s. Th
e ai
m o
f thi
s pap
er is
to sh
ow th
e pr
oper
ties o
f Nan
obod
ies,
thei
r pro
duct
ion
and
expr
essio
n, a
pplic
atio
ns a
nd th
eir c
linic
al st
atus
.
Deff
ar e
t al.
2009
Bio
tech
nolo
gica
l ap
plic
atio
nsSe
lect
ion
syst
ems
Cam
elid
s pro
duce
func
tiona
l ant
ibod
ies d
evoi
d of
ligh
t cha
ins o
f whi
ch th
e si
ngle
N-t
erm
inal
dom
ain
is fu
lly c
apab
le o
f ant
igen
bi
ndin
g. Th
ese
sing
le-d
omai
n an
tibod
y fr
agm
ents
(VH
Hs o
r Nan
obod
ies)
hav
e se
vera
l adv
anta
ges f
or b
iote
chno
logi
cal a
pplic
a-tio
ns. Th
ey a
re w
ell e
xpre
ssed
in m
icro
orga
nism
s and
hav
e a
high
stab
ility
and
solu
bilit
y. Fu
rthe
rmor
e, th
ey a
re w
ell s
uite
d fo
r co
nstr
uctio
n of
larg
er m
olec
ules
and
sele
ctio
n sy
stem
s suc
h as
pha
ge, y
east
, or r
ibos
ome
disp
lay.
This
min
irev
iew
offe
rs a
n ov
er-
view
of t
heir
pro
pert
ies a
s com
pare
d to
con
vent
iona
l ant
ibod
ies,
thei
r pro
duct
ion
in m
icro
orga
nism
s, w
ith a
focu
s on
yeas
ts, a
nd
thei
r the
rape
utic
app
licat
ions
.
Har
mse
n an
d de
H
aard
200
7
Ant
igen
-bin
ding
sit
eM
utat
ions
VH
-VL
pair
Nov
el e
pito
pes
The
antig
en-b
indi
ng si
te o
f ant
ibod
ies f
rom
ver
tebr
ates
is fo
rmed
by
com
bini
ng th
e va
riab
le d
omai
ns o
f a h
eavy
cha
in (V
H) a
nd
a lig
ht c
hain
(VL)
. How
ever
, ant
ibod
ies f
rom
cam
els a
nd Il
amas
are
an
impo
rtan
t exc
eptio
n to
this
in th
at th
eir s
era
cont
ain,
in
addi
tion,
a u
niqu
e ki
nd o
f ant
ibod
y th
at is
form
ed b
y he
avy
chai
ns o
nly
The
antig
en-b
indi
ng si
te o
f the
se a
ntib
odie
s con
sist
s of
one
sing
le d
omai
n, re
ferr
ed to
as V
HH
. This
art
icle
revi
ews t
he m
utat
ions
and
stru
ctur
al a
dapt
atio
ns th
at h
ave
ta k
en p
lace
to
resh
ape
a V
H o
f a V
H-V
L pa
ir in
to a
sing
le-d
omai
n V
HH
with
rete
ntio
n of
a su
ffici
ent v
aria
bilit
y, Th
e V
HH
has
a p
oten
t ant
igen
-bi
ndin
g ca
paci
ty a
nd p
rovi
des t
he a
dvan
tage
of i
nter
actin
g w
ith n
ovel
epi
tope
s tha
t are
inac
cess
ible
to c
onve
ntio
nal V
H-V
L pa
irs.
Muy
lder
-m
ans e
t al.
2001
Can
cer
ther
apeu
tics
Ove
r the
yea
rs, m
any
antib
odie
s hav
e be
en su
cces
sful
ly g
ener
ated
to tr
eat p
atie
nts w
ith li
fe-t
hrea
teni
ng d
isea
ses,
mos
t not
ably
ca
ncer
. Whi
le th
e fir
st g
ener
atio
n of
ant
ibod
ies,
orig
inat
ing
from
mic
e, c
ause
d se
vere
side
effe
cts a
nd w
ere
rela
tivel
y in
effici
ent,
tech
nolo
gica
l adv
ance
s hav
e m
ade
it po
ssib
le to
obt
ain
fully
hum
an a
ntib
odie
s for
ther
apeu
tic u
se. ‘
Hea
vy-c
hain
onl
y’ a
ntib
odie
s ha
ve re
cent
ly b
een
disc
over
ed in
the
bloo
d of
cam
elid
s. Be
caus
e of
thei
r siz
e, th
e an
tigen
-bin
ding
uni
ts o
f the
se a
ntib
odie
s com
pris-
ing
only
a si
ngle
Ig fo
ld a
re c
alle
d N
anob
odie
s. Th
ese
antib
ody
frag
men
ts h
ave
seve
ral r
emar
kabl
e fe
atur
es th
at m
ake
them
idea
l ca
ndid
ates
as n
ext-
gene
ratio
n ca
ncer
ther
apeu
tics.
Part
icul
arly
app
ealin
g is
thei
r abi
lity
to si
mul
tane
ously
inhi
bit v
ario
us c
ruci
al
grow
th fa
ctor
rece
ptor
s or t
heir
ligan
ds w
ith a
sing
le m
olec
ule.
In a
dditi
on, t
hey
are
easy
to c
lone
and
exp
ress
on
the
tip o
f fila
men
-to
us p
hage
, whi
ch o
pens
the
poss
ibili
ty to
sele
ct fo
r Nan
obod
ies i
nduc
ing
part
icul
ar b
iolo
gica
l effe
cts.
Nan
obod
ies h
ave
pote
ntia
l to
beco
me
impo
rtan
t can
cer t
hera
peut
ics i
n th
e ne
ar fu
ture
, dis
play
ing
uneq
ualle
d an
d un
prec
eden
ted
effica
cies
in tr
eatm
ent.
Roov
ers e
t al
. 200
7
Stru
ctur
al p
rope
rtie
sTh
erap
euti
c ap
plic
atio
nC
linic
al tr
ials
ALX
-008
1
Nan
obod
ies a
re th
erap
eutic
pro
tein
s der
ived
from
the
heav
y-ch
ain
vari
able
(V(H
)H) d
omai
ns th
at o
ccur
nat
ural
ly in
hea
vy-c
hain
-on
ly Ig
mol
ecul
es in
cam
elid
ae. Th
ese
V(H
)H d
omai
ns a
re th
e sm
alle
st k
now
n an
tigen
-bin
ding
ant
ibod
y fr
agm
ents
. Nan
obod
ies
can
be e
asily
pro
duce
d in
pro
kary
otic
or e
ukar
yotic
hos
t org
anis
ms,
and
thei
r uni
que
biop
hysi
cal a
nd p
harm
acol
ogic
al c
hara
cter
-is
tics r
ende
r the
se m
olec
ules
idea
l can
dida
tes f
or d
rug
deve
lopm
ent.
This
revi
ew d
escr
ibes
the
stru
ctur
al p
rope
rtie
s of n
anob
odie
s an
d fo
cuse
s on
thei
r uni
que
feat
ures
, whi
ch d
istin
guis
hes t
hese
mol
ecul
es fr
om o
ther
ant
ibod
y fo
rmat
s and
smal
l-mol
ecul
e dr
ugs.
Poss
ible
ther
apeu
tic a
pplic
atio
ns o
f nan
obod
ies a
re d
iscu
ssed
and
dat
a fr
om p
hase
I cl
inic
al tr
ials
of t
heno
vel ‘
first
-in-
clas
s’ an
ti-th
rom
botic
age
nt A
LX-0
081
(Abl
ynx
NV
) are
pre
sent
ed.
Van
Bock
-st
aele
et a
l. 20
09
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
448
Ther
apeu
tic
appl
icat
ions
Viru
ses
In 1
989,
a n
ew ty
pe o
f ant
ibod
y w
as id
entifi
ed, fi
rst i
n th
e se
ra o
f dro
med
arie
s and
late
r als
o in
all
othe
r spe
cies
of t
he C
amel
idae
fa
mily
. Thes
e an
tibod
ies d
o no
t con
tain
a li
ght c
hain
and
als
o la
ck th
e fir
st c
onst
ant h
eavy
dom
ain.
Tod
ay it
is st
ill u
ncle
ar w
hat
the
evol
utio
nary
adv
anta
ge o
f suc
h he
avy
chai
n-on
ly a
ntib
odie
s cou
ld b
e. In
shar
p co
ntra
st, t
he b
road
app
licab
ility
of t
he is
olat
ed
vari
able
ant
igen
-bin
ding
dom
ains
(VH
H) w
as ra
pidl
y re
cogn
ized
, esp
ecia
lly fo
r the
dev
elop
men
t of t
hera
peut
ic p
rote
ins,
cal
led
Nan
obod
ies.
Her
e w
e su
mm
ariz
e fir
st so
me
of th
e un
ique
cha
ract
eris
tics a
nd fe
atur
es o
f VH
Hs.
Thes
e w
ill n
ext b
e de
scri
bed
in
the
cont
ext o
f diff
eren
t exp
erim
enta
l the
rape
utic
app
licat
ions
of N
anob
odie
s aga
inst
diff
eren
t vir
uses
: HIV
, Hep
atiti
s B v
irus
, in
fluen
za v
irus
, Res
pira
tory
Syn
cytia
l vir
us, R
abie
s vir
us, F
MD
V, P
olio
viru
s, R
otav
irus
, and
PER
Vs. N
ext,
the
diag
nost
ic a
pplic
a-tio
n of
VH
Hs (
Vacc
inia
vir
us, M
arbu
rg v
irus
and
pla
nt T
ulip
vir
us X
), as
wel
l as a
n in
dust
rial
app
licat
ion
(lytic
lact
ococ
cal 9
36
phag
e) w
ill b
e de
scri
bed.
In a
dditi
on, t
he d
escr
ibed
dat
a sh
ow th
at m
onov
alen
t Nan
obod
ies c
an p
osse
ss u
niqu
e ch
arac
teri
stic
s not
ob
serv
ed w
ith c
onve
ntio
nal a
ntib
odie
s. Th
e st
raig
htfo
rwar
d fo
rmat
ting
into
biv
alen
t, m
ultiv
alen
t, an
d/or
mul
tispe
cific
Nan
obod
-ie
s allo
wed
tailo
ring
mol
ecul
es fo
r pot
ency
and
cro
ss-r
eact
ivity
aga
inst
vir
al ta
rget
s with
hig
h se
quen
ce d
iver
sity
.
Vanl
and-
scho
ot e
t al
. 201
1
VH
HV
NA
RIm
mun
e fu
ncti
on
mod
ulat
ing
Toxi
n/m
icro
be ta
rget
ing
Ant
ibod
ies a
re im
port
ant t
ools
for e
xper
imen
tal r
esea
rch
and
med
ical
app
licat
ions
. Mos
t ant
ibod
ies a
re c
ompo
sed
of tw
o he
avy
and
two
light
cha
ins.
Bot
h ch
ains
con
trib
ute
to th
e an
tigen
-bin
ding
site
whi
ch is
usu
ally
flat
or c
onca
ve. I
n ad
ditio
n to
thes
e co
nven
tiona
l ant
ibod
ies,
llam
as, o
ther
cam
elid
s, a
nd sh
arks
als
o pr
oduc
e an
tibod
ies c
ompo
sed
only
of h
eavy
cha
ins.
The
antig
en-
bind
ing
site
of t
hese
unu
sual
hea
vy c
hain
ant
ibod
ies (
hcA
bs) i
s for
med
onl
y by
a si
ngle
dom
ain,
des
igna
ted
VH
H in
cam
elid
hcA
bs
and
VN
AR
in sh
ark
hcA
bs. V
HH
and
VN
AR
are
easi
ly p
rodu
ced
as re
com
bina
nt p
rote
ins,
des
igna
ted
sing
le d
omai
n an
tibod
ies
(sdA
bs) o
r nan
obod
ies.
The
CD
R3 re
gion
of t
hese
sdA
bs p
osse
sses
the
extr
aord
inar
y ca
paci
ty to
form
long
fing
erlik
e ex
tens
ions
th
at c
an e
xten
d in
to c
aviti
es o
n an
tigen
s, e
.g.,
the
activ
e si
te c
revi
ce o
f enz
ymes
. Oth
er a
dvan
tage
ous f
eatu
res o
f nan
obod
ies
incl
ude
thei
r sm
all s
ize,
hig
h so
lubi
lity,
ther
mal
stab
ility
, ref
oldi
ng c
apac
ity, a
nd g
ood
tissu
e pe
netr
atio
n in
viv
o. H
ere
we
revi
ew
the
resu
lts o
f sev
eral
rece
nt p
roof
-of-
prin
cipl
e st
udie
s tha
t ope
n th
e ex
citin
g pe
rspe
ctiv
e of
usi
ng sd
Abs
for m
odul
atin
g im
mun
e fu
nctio
ns a
nd fo
r tar
getin
g to
xins
and
mic
robe
s.
Wes
o-lo
wsk
i et a
l. 20
09
Imm
unos
enso
rIm
mun
oaffi
nity
ch
rom
atog
raph
yIm
mun
o-im
agin
g
With
the
adve
nt o
f new
ant
ibod
y en
gine
erin
g te
chno
logi
es, c
onve
ntio
nal a
ntib
odie
s hav
e be
en m
inim
ized
into
smal
ler a
ntib
ody
form
ats.
Smal
l siz
e is
an im
port
ant a
dvan
tage
for c
urre
nt a
nd fu
ture
dia
gnos
tic d
evel
opm
ent.
Nan
obod
ies (
Abl
ynx)
are
am
ong
the
smal
lest
kn
own
antig
en-b
indi
ng a
ntib
ody
frag
men
ts, a
nd a
re d
eriv
ed fr
om th
e he
avy-
chai
n on
ly a
ntib
odie
s tha
t occ
ur n
atur
ally
in th
e se
rum
of
Cam
elid
ae. E
ndow
ed b
y na
tura
l evo
lutio
n, th
ese
Nan
obod
ies i
nher
ently
exh
ibit
uniq
ue b
ioph
ysic
al, b
ioch
emic
al a
nd p
harm
acol
ogic
al
char
acte
ristic
s. In
add
ition
to th
eir e
xcel
lent
pot
entia
l as m
olec
ules
in d
rug
deve
lopm
ent,
Nan
obod
ies p
osse
ss v
ery
attr
activ
e fu
nctio
nal
prop
ertie
s tha
t aid
in th
eir d
evel
opm
ent f
or d
iagn
ostic
tool
s. H
ere
we
pres
ent s
ever
al e
xam
ples
of c
urre
ntly
ava
ilabl
e ap
plic
atio
ns o
f N
anob
odie
s to
the
field
of i
mm
unos
enso
r for
can
cer,
imm
unoa
ffini
ty c
hrom
atog
raph
y, in
viv
o an
d in
trac
ellu
lar i
mag
ing.
Hua
ng e
t al
. 201
0
Bio
tech
nolo
gica
l ap
plic
atio
nsTh
e di
scov
ery
of th
e si
ngle
-dom
ain
antib
ody’s
pot
entia
ls h
as st
imul
ated
thei
r use
in a
n in
crea
sing
var
iety
of fi
elds
. The
rapi
d ac
cum
ulat
ion
of a
rtic
les d
escr
ibin
g ne
w a
pplic
atio
ns a
nd fu
rthe
r dev
elop
men
ts o
f est
ablis
hed
appr
oach
es h
as m
ade
it, th
eref
ore,
ne
cess
ary
to u
pdat
e th
e pr
evio
us re
view
s with
a n
ew a
nd m
ore
com
plet
e su
mm
ary
of th
e to
pic.
Con
clus
ions
: Bes
ide
the
nece
ssar
y ta
sk o
f upd
atin
g, th
is w
ork
anal
yses
in d
etai
l som
e ap
plic
ativ
e as
pect
s of t
he si
ngle
-dom
ain
antib
odie
s tha
t hav
e be
en o
vers
een
in th
e pa
st, s
uch
as th
eir e
ffica
cy in
affi
nity
chr
omat
ogra
phy,
as c
o-cr
ysta
lliza
tion
chap
eron
es, p
rote
in a
ggre
gatio
n co
ntro
llers
, en
zym
e ac
tivity
tune
rs, a
nd th
e sp
ecifi
citie
s of t
he u
ncon
vent
iona
l sin
gle-
dom
ain
frag
men
ts.
de M
arco
20
11
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
449
Imm
uno-
imag
ing
Imm
uno-
imag
ing
is a
dev
elop
ing
tech
nolo
gy th
at a
ims a
t stu
dyin
g di
seas
e in
pat
ient
s usi
ng im
agin
g te
chni
ques
such
as p
ositr
on
emis
sion
tom
ogra
phy
in c
ombi
natio
n w
ith ra
diol
abel
ed im
mun
oglo
bulin
der
ived
targ
etin
g pr
obes
. Nan
obod
ies a
re th
e sm
alle
st
antig
en-b
indi
ng a
ntib
ody-
frag
men
ts a
nd sh
ow fa
st a
nd sp
ecifi
c ta
rget
ing
in v
ivo.
Thes
e pr
obes
are
cur
rent
ly u
nder
inve
stig
atio
n as
th
erap
eutic
s but
pre
clin
ical
stud
ies i
ndic
ate
that
nan
obod
ies c
ould
als
o be
com
e th
e ne
xt g
ener
atio
n of
mag
ic b
ulle
ts fo
r im
mun
o-im
agin
g. In
itial
dat
a sh
ow th
at im
agin
g ca
n be
per
form
ed a
s ear
ly a
s one
hou
r pos
t-in
ject
ion
enab
ling
the
use
of sh
ort-
lived
radi
o-is
otop
es. Th
ese
uniq
ue p
rope
rtie
s sho
uld
enab
le p
atie
nt fr
iend
ly a
nd sa
fe im
agin
g pr
otoc
ols.
This
revi
ew fo
cuse
s on
the
curr
ent
stat
us o
f rad
iola
bele
d na
nobo
dies
as t
arge
ting
prob
es fo
r im
mun
o-im
agin
g.
Vane
ycke
n et
al.
2011
a
Man
-mad
e co
njug
ate
Ant
ibod
ies a
re la
rge
and
com
plex
mol
ecul
es, w
ith tw
o id
entic
al p
arts
that
bin
d in
depe
nden
tly o
f eac
h ot
her o
nto
the
antig
en a
nd th
e th
ird p
art o
f the
mol
ecul
e th
at d
icta
tes t
he e
ffect
or fu
nctio
n(s)
. To
impr
ove
the
ther
apeu
tic v
alue
of a
ntib
odie
s, pr
otei
n-en
gine
erin
g en
deav
ors r
educ
ed th
e si
ze o
f the
ant
igen
-bin
ding
moi
ety
to a
sing
le-d
omai
n un
it. O
ccas
iona
lly, i
t was
dem
onst
rate
d th
at th
e si
ngle
-do
mai
n an
tigen
-bin
ding
der
ivat
ives
of a
ntib
odie
s can
hav
e –
on th
eir o
wn
– an
ago
nist
ic (o
r ant
agon
istic
) effe
ct o
n th
eir t
arge
t. Th
e sm
all s
ize
and
stri
ct m
onom
eric
beh
avio
r, in
com
bina
tion
with
oth
er b
ioch
emic
al p
rope
rtie
s suc
h as
hig
h so
lubi
lity
and
high
spec
ific-
ity a
nd a
ffini
ty fo
r the
cog
nate
ant
igen
, mak
e si
ngle
-dom
ain
antib
odie
s ide
al to
des
ign
nove
l man
-mad
e co
njug
ates
har
ness
ed w
ith
inno
vativ
e eff
ecto
r fun
ctio
ns o
utsi
de th
e re
ach
of c
lass
ical
ant
ibod
ies.
Saer
ens e
t al
. 200
8
Smal
ler
reco
mbi
nant
an
tibo
dy fr
agm
ents
Mul
tiva
lent
/mul
tisp
ecifi
c re
agen
tSi
ngle
ant
ibod
y do
mai
nsEn
hanc
ed th
erap
euti
c effi
cacy
With
18
mon
oclo
nal a
ntib
ody
(mA
b) p
rodu
cts c
urre
ntly
on
the
mar
ket a
nd m
ore
than
100
in c
linic
al tr
ials
, it i
s cle
ar th
at e
ngi-
neer
ed a
ntib
odie
s hav
e co
me
of a
ge a
s bio
phar
mac
eutic
als.
In fa
ct, b
y 20
08, e
ngin
eere
d an
tibod
ies a
re p
redi
cted
to a
ccou
nt fo
r >
30%
of a
ll re
venu
es in
the
biot
echn
olog
y m
arke
t. Sm
alle
r rec
ombi
nant
ant
ibod
y fr
agm
ents
(for
exa
mpl
e, c
lass
ic m
onov
alen
t an
tibod
y fr
agm
ents
(Fab
, scF
v)) a
nd e
ngin
eere
d va
rian
ts (d
iabo
dies
, tri
abod
ies,
min
ibod
ies a
nd si
ngle
-dom
ain
antib
odie
s) a
re
now
em
ergi
ng a
s cre
dibl
e al
tern
ativ
es. Th
ese
frag
men
ts re
tain
the
targ
etin
g sp
ecifi
city
of w
hole
mA
bs b
ut c
an b
e pr
oduc
ed m
ore
econ
omic
ally
and
pos
sess
oth
er u
niqu
e an
d su
peri
or p
rope
rtie
s for
a ra
nge
of d
iagn
ostic
and
ther
apeu
tic a
pplic
atio
ns. A
ntib
ody
frag
men
ts h
ave
been
forg
ed in
to m
ultiv
alen
t and
mul
tispe
cific
reag
ents
, lin
ked
to th
erap
eutic
pay
load
s (su
ch a
s rad
ionu
clid
es,
toxi
ns, e
nzym
es, l
ipos
omes
and
vir
uses
) and
eng
inee
red
for e
nhan
ced
ther
apeu
tic e
ffica
cy. R
ecen
tly, s
ingl
e an
tibod
y do
mai
ns h
ave
been
eng
inee
red
and
sele
cted
as t
arge
ting
reag
ents
aga
inst
hith
erto
imm
unos
ilent
cav
ities
in e
nzym
es, r
ecep
tors
and
infe
ctio
us
agen
ts. S
ingl
e-do
mai
n an
tibod
ies a
re a
ntic
ipat
ed to
sign
ifica
ntly
exp
and
the
repe
rtoi
re o
f ant
ibod
y-ba
sed
reag
ents
aga
inst
the
vast
ra
nge
of n
ovel
bio
mar
kers
bei
ng d
isco
vere
d th
roug
h pr
oteo
mic
s. A
s thi
s rev
iew
aim
s to
show
, the
re is
trem
endo
us p
oten
tial f
or a
ll an
tibod
y fr
agm
ents
eith
er a
s rob
ust d
iagn
ostic
reag
ents
(for
exa
mpl
e in
bio
sens
ors)
, or a
s non
imm
unog
enic
in v
ivo
biop
harm
a-ce
utic
als w
ith su
peri
or b
iodi
stri
butio
n an
d bl
ood
clea
ranc
e pr
oper
ties.
Hol
liger
an
d H
udso
n 20
05
Mol
ecul
ar im
agin
gTa
rget
-ori
ente
d th
erap
ies
Ant
ibod
y de
riva
tive
Alt
erna
tive
pro
tein
The
rapi
d an
d on
goin
g di
scov
ery
of n
ew d
isea
se re
late
d bi
omar
kers
lead
s to
a dr
amat
ic p
arad
igm
cha
nge
in h
uman
hea
lthca
re a
nd
cons
titut
es th
e ba
sis f
or a
trul
y pe
rson
aliz
ed m
edic
ine.
Mol
ecul
ar im
agin
g en
able
s ear
ly d
etec
tion
and
clas
sific
atio
n of
hum
an d
isea
ses
and
prov
ides
val
uabl
e da
ta fo
r opt
imiz
ed, t
arge
t-or
ient
ed th
erap
ies.
By n
ow, t
he b
ioch
emic
al a
nd p
hysi
olog
ical
pro
pert
ies o
f ant
ibod
y de
riva
tives
or a
ltern
ativ
e pr
otei
n sc
affol
ds c
an b
e en
gine
ered
for t
he d
etec
tion
of a
wid
e ra
nge
of ta
rget
stru
ctur
es. Th
e su
cces
sful
ap
plic
atio
n of
thes
e re
agen
ts in
ani
mal
s, xe
nogr
aft m
odel
s and
cel
ls in
pre
clin
ical
rese
arch
cle
arly
dem
onst
rate
thei
r util
ity fo
r mol
ecu-
lar i
mag
ing.
Des
pite
thes
e pr
omis
ing
pers
pect
ives
, onl
y a
few
ant
ibod
ies a
nd re
com
bina
nt p
rote
ins a
re u
sed
yet f
or m
olec
ular
imag
-in
g in
hum
an m
edic
ine.
Esp
ecia
lly th
e hi
gh sa
fety
dem
ands
and
the
need
to e
limin
ate
off ta
rget
effe
cts i
n hu
man
s req
uire
ext
ensi
ve
rese
arch
and
dev
elop
men
t effo
rts.
Rom
er e
t al
. 201
1
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
450
X-r
ay c
ryst
allo
grap
hyC
ryst
alliz
atio
n
chap
eron
eC
onfo
rmat
iona
l he
tero
gene
ity
The
prep
arat
ion
of d
iffra
ctio
n qu
ality
cry
stal
s rem
ains
the
maj
or b
ottle
neck
in m
acro
mol
ecul
ar X
-ray
cry
stal
logr
aphy
. A c
ryst
alliz
a-tio
n ch
aper
one
is a
n au
xilia
ry p
rote
in, s
uch
as fr
agm
ents
of m
onoc
lona
l ant
ibod
ies,
that
bin
ds to
and
incr
ease
s the
cry
stal
lizat
ion
prob
abili
ty o
f a ta
rget
mol
ecul
e of
inte
rest
. Suc
h ch
aper
ones
redu
ce c
onfo
rmat
iona
l het
erog
enei
ty, m
ask
coun
terp
rodu
ctiv
e su
rfac
es
whi
le e
xten
ding
surf
aces
pre
disp
osed
to fo
rmin
g cr
ysta
l con
tact
s, a
nd p
rovi
de p
hasi
ng in
form
atio
n. C
ryst
alliz
atio
n ch
aper
ones
gen
-er
ated
usi
ng re
com
bina
nt te
chno
logi
es h
ave
emer
ged
as su
peri
or a
ltern
ativ
es th
at in
crea
se th
e th
roug
hput
and
elim
inat
e in
here
nt
limita
tions
ass
ocia
ted
with
ant
ibod
y pr
oduc
tion
by a
nim
al im
mun
izat
ion
and
the
hybr
idom
a te
chno
logy
.
Koi
de 2
009
Rec
entl
y pu
blis
hed
m
onog
raph
Sing
le D
omai
n A
ntib
odie
s
http
://w
ww.
spri
nger
.com
/bio
med
/imm
unol
ogy/
book
/978
-1-6
1779
-967
-9Th
e de
velo
pmen
t of t
he h
ybri
dom
a te
chno
logy
cre
ated
the
poss
ibili
ty to
obt
ain
unlim
ited
amou
nts o
f mon
oclo
nal a
ntib
odie
s (m
Ab)
w
ith h
igh
spec
ifici
ty a
nd a
ffini
ty fo
r any
targ
et a
nd to
intr
oduc
e m
Abs
in a
wid
e ra
nge
of a
pplic
atio
ns. E
xam
ples
of a
ntib
ody-
base
d dr
ugs i
n th
erap
eutic
sett
ings
and
ant
ibod
y-ba
sed
prob
es in
dia
gnos
tics a
re in
finite
. How
ever
, the
bul
ky si
ze o
f mA
bs, c
ostly
pro
duc-
tion,
and
cum
bers
ome
engi
neer
ing
reta
rded
or h
ampe
red
regu
larly
thei
r str
eam
lined
dev
elop
men
t in
som
e ap
plic
atio
ns. C
onse
quen
tly,
mA
bs b
ecam
e th
e fo
cus o
f man
y at
tem
pts t
o m
inim
ize
the
size
and
com
plex
ity o
f the
ir an
tigen
-bin
ding
frag
men
ts. E
vent
ually
, the
se
effor
ts le
d to
the
reco
mbi
nant
pro
duct
ion
of sm
alle
r ant
igen
bind
ing
frag
men
ts su
ch a
s Fab
or s
cFv
(whe
re a
synt
hetic
link
er c
onne
cts
the
vari
able
dom
ains
of h
eavy
and
ligh
t cha
in, i
.e.,
VH
and
VL)
, and
eve
n sd
Abs
(sin
gle
dom
ain
antib
odie
s der
ived
mos
tly o
f the
VH
). A
lthou
gh th
e fir
st se
t of s
dAbs
offe
red
sign
ifica
nt a
dvan
tage
s, th
ey a
lso
suffe
red
from
mul
tiple
shor
tcom
ings
, all
of w
hich
hav
e be
en
rem
edia
ted
by e
lega
nt e
ngin
eeri
ng. I
nter
estin
gly,
whi
le sc
ient
ists
wer
e de
sign
ing,
eng
inee
ring
, and
shap
ing
the
idea
l sdA
b, a
sere
ndip
i-to
us d
isco
very
show
ed th
at a
sim
ilar e
ngin
eeri
ng o
ccur
red
alre
ady
in n
atur
e in
the
cam
elid
s, an
d la
ter o
n, it
was
foun
d th
at c
artil
agi-
nous
fish
ant
ibod
ies p
erfo
rmed
the
exer
cise
eve
n ea
rlier
on
in e
volu
tion.
Thes
e an
imal
s hav
e in
thei
r blo
od fu
nctio
nal a
ntib
ody
isot
ype
com
pose
d of
hea
vy c
hain
s – o
nly
that
lack
ligh
t cha
ins,
in a
dditi
on to
the
clas
sica
l ant
ibod
ies c
onta
inin
g tw
o he
avy
and
two
light
ch
ains
. Thes
e he
avy-
chai
n an
tibod
ies (
HC
Abs
) rec
ogni
ze th
e an
tigen
via
a si
ngle
var
iabl
e do
mai
n, re
ferr
ed to
as V
HH
or V
-NA
R. Th
e V
HH
or V
-NA
R is
the
smal
lest
inta
ct a
ntig
en-b
indi
ng fr
agm
ent t
hat c
an b
e pr
oduc
ed re
com
bina
ntly
at l
ow c
ost.
The
valu
able
pro
per-
ties o
f man
-mad
e sd
Abs
, VH
Hs,
and
V-N
ARs
incl
udin
g so
lubi
lity
and
stab
ility
, hig
h affi
nity
and
spec
ifici
ty fo
r the
ir co
gnat
e an
tigen
, sm
all s
ize
and
stri
ct m
onom
eric
beh
avio
r offe
r man
y op
port
uniti
es. A
s a re
sult,
seve
ral s
pin-
off c
ompa
nies
hav
e be
en fo
unde
d in
Aus
-tr
alia
, Bel
gium
, Eng
land
, Ger
man
y, N
ethe
rland
s, an
d Sc
otla
nd th
at in
trod
uced
thes
e pr
otei
ns su
cces
sful
ly in
a w
ide
rang
e of
app
lica-
tions
to c
over
a sp
ecia
l nee
d in
rese
arch
or e
ven
to p
rodu
ce n
ext-
gene
ratio
n th
erap
eutic
s in
the
clin
ic (P
refa
ce b
y th
e ed
itors
).
Title
s of t
he c
ontr
ibut
ions
:PA
RT I
OV
ERV
IEW
OF
SIN
GLE
DO
MA
IN A
NT
IBO
DIE
SFr
om W
hole
Mon
oclo
nal A
ntib
odie
s to
Sing
le D
omai
n A
ntib
odie
s: Th
ink
Smal
lIn
trod
uctio
n to
Hea
vy C
hain
Ant
ibod
ies a
nd D
eriv
ed N
anob
odie
s O
verv
iew
and
Dis
cove
ry o
f IgN
ARs
and
Gen
erat
ion
of V
NA
RsPA
RT II
SIN
GLE
DO
MA
IN A
NT
IBO
DY
LIBR
ARY
CO
NST
RUC
TIO
NC
reat
ion
of th
e La
rge
and
Hig
hly
Func
tiona
l Syn
thet
ic R
eper
toire
of H
uman
VH
and
Vκ
Dom
ain
Ant
ibod
ies
Prep
arat
ion
of a
Naï
ve L
ibra
ry o
f Cam
elid
Sin
gle
Dom
ain
Ant
ibod
ies
PART
III S
ELEC
TIO
N O
F SI
NG
LE D
OM
AIN
AN
TIB
OD
IES
Sele
ctio
n by
Pha
ge D
ispl
ay o
f Sin
gle
Dom
ain
Ant
ibod
ies S
peci
fic to
Ant
igen
s in
Thei
r Nat
ive
Con
form
atio
nSe
mia
utom
ated
Pan
ning
of N
aive
Cam
elid
ae L
ibra
ries
and
Sel
ectio
n of
Sin
gle-
Dom
ain
Ant
ibod
ies A
gain
st P
eptid
e A
ntig
ens
Pich
ia S
urfa
ce D
ispl
ay: A
Too
l for
Scr
eeni
ng S
ingl
e D
omai
n A
ntib
odie
sBa
cter
ial T
wo
Hyb
rid:
A V
ersa
tile
One
-Ste
p In
trac
ellu
lar S
elec
tion
Met
hod
Intr
acel
lula
r Ant
ibod
y C
aptu
re (I
AC
) Met
hods
or S
ingl
e D
omai
n A
ntib
odie
sSe
lect
ion
of F
unct
iona
l Sin
gle
Dom
ain
Ant
ibod
y Fr
agm
ents
for I
nter
feri
ng w
ith P
rote
in–P
rote
in In
tera
ctio
ns In
side
Cel
ls: A
“One
Pl
asm
id” M
amm
alia
n Tw
o-H
ybri
d Sy
stem
Saer
ens
and
Muy
l-de
rman
s 20
12
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
451
Cel
l-Fre
e Se
lect
ion
of D
omai
n A
ntib
odie
s by
In V
itro
Com
part
men
taliz
atio
n Se
lect
ion
of V
HH
s Und
er A
pplic
atio
n C
ondi
tions
Isol
atio
n an
d C
hara
cter
izat
ion
of C
lost
ridi
um d
iffici
le T
oxin
-Spe
cific
Sin
gle-
Dom
ain
Ant
ibod
ies
Sele
ctio
n of
VH
H A
ntib
ody
Frag
men
ts Th
at R
ecog
nize
Diff
eren
t Aβ
Dep
ositi
ons U
sing
Com
plex
Imm
une
Libr
arie
sPA
RT IV
EX
PRES
SIO
N O
F SI
NG
LE D
OM
AIN
AN
TIB
OD
IES
AN
D D
ERIV
ATIV
ESEx
pres
sion
of S
ingl
e-D
omai
n A
ntib
odie
s in
Bact
eria
l Sys
tem
sEx
pres
sion
of V
HH
s in
Sacc
haro
myc
es c
erev
isia
eSt
able
Exp
ress
ion
of C
him
eric
Hea
vy C
hain
Ant
ibod
ies i
n C
HO
Cel
lsPr
oduc
tion
of C
amel
-Lik
e A
ntib
odie
s in
Plan
tsPA
RT V
IMPR
OV
EMEN
T A
ND
APP
LIC
ATIO
NS
OF
SIN
GLE
DO
MA
IN A
NT
IBO
DIE
SSe
lect
ing
and
Puri
fyin
g A
uton
omou
s Hum
an V
aria
ble
Hea
vy (V
H) D
omai
nsSo
lubi
lity
and
Stab
ility
Eng
inee
ring
of H
uman
VH
Dom
ains
Impr
ovem
ent o
f Pro
teol
ytic
Sta
bilit
y Th
roug
h In
Sili
co E
ngin
eeri
ngSe
lect
ion
of H
uman
VH
Sin
gle
Dom
ains
with
Impr
oved
Bio
phys
ical
Pro
pert
ies b
y Ph
age
Dis
play
Im
prov
emen
t of S
ingl
e D
omai
n A
ntib
ody
Stab
ility
by
Dis
ulfid
e Bo
nd In
trod
uctio
nC
hara
cter
izat
ion
of S
ingl
e-D
omai
n A
ntib
odie
s with
an
Engi
neer
ed D
isul
fide
Bond
Affi
nity
Mat
urat
ion
of S
ingl
e-D
omai
n A
ntib
odie
s by
Yeas
t Sur
face
Dis
play
Mul
tival
ent D
ispl
ay o
f Sin
gle-
Dom
ain
Ant
ibod
ies
Met
hods
for D
eter
min
ing
the
PK P
aram
eter
s of A
lbud
Abs
and
of L
ong
Seru
m H
alf-
Life
Dru
gs M
ade
Usi
ng th
e A
lbud
Ab
Tech
nolo
gyFl
uore
scen
t Pro
tein
Spe
cific
Nan
otra
ps to
Stu
dy P
rote
in–P
rote
in In
tera
ctio
ns a
nd H
isto
ne-T
ail P
eptid
e Bi
ndin
gSi
te-S
peci
fic L
abel
ing
of H
is-T
agge
d N
anob
odie
s with
99m
Tc: A
Pra
ctic
al G
uide
N
anob
ody-
Base
d C
hrom
atin
Imm
unop
reci
pita
tion
Use
r-Fr
iend
ly E
xpre
ssio
n Pl
asm
ids E
nabl
e th
e Fu
sion
of V
HH
s to
App
licat
ion-
Spec
ific
Tags
App
licat
ion
of S
ingl
e-D
omai
n A
ntib
odie
s in
Tum
or H
isto
chem
istr
yPA
RT V
I CA
SE S
TU
DIE
SN
anob
odie
s as S
truc
tura
l Pro
bes o
f Pro
tein
Mis
fold
ing
and
Fibr
il Fo
rmat
ion
Mol
ecul
ar Im
agin
g U
sing
Nan
obod
ies:
A C
ase
Stud
yC
ase
Stud
y on
Liv
e C
ell A
popt
osis
-Ass
ay U
sing
Lam
in-C
hrom
obod
y C
ell-L
ines
for H
igh-
Con
tent
Ana
lysi
s
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
452
Tabl
e 3.
Cam
elid
and
sha
rk h
eavy
-cha
in a
ntib
odie
s
Hea
vy-c
hain
dim
erC
amel
us d
rom
edar
ius
Ant
igen
-bin
ding
re
pert
oire
CH
1 do
mai
nA
ntib
ody
engi
neer
ing
Ran
dom
ass
ocia
tion
of V
L an
d V
H r
eper
toir
es c
ontr
ibut
es c
onsi
dera
bly
to a
ntib
ody
dive
rsit
y. T
he d
iver
sity
and
the
affin
ity
are
then
incr
ease
d by
hyp
erm
utat
ion
in B
cel
ls lo
cate
d in
ger
min
al c
entr
es. E
xcep
t in
the
case
of ‘
heav
y ch
ain’
dis
ease
, nat
u-ra
lly o
ccur
ring
hea
vy-c
hain
ant
ibod
ies
have
not
bee
n de
scri
bed,
alth
ough
ant
igen
bin
ding
has
bee
n de
mon
stra
ted
for
sepa
-ra
ted
heav
y ch
ains
or
clon
ed V
H d
omai
ns. H
ere
we
inve
stig
ate
the
pres
ence
of c
onsi
dera
ble
amou
nts
of Ig
G-l
ike
mat
eria
l of
M(r
) 100
K in
the
seru
m o
f the
cam
el (C
amel
us d
rom
edar
ius)
. The
se m
olec
ules
are
com
pose
d of
hea
vy-c
hain
dim
ers
and
are
devo
id o
f lig
ht c
hain
s, b
ut n
ever
thel
ess
have
an
exte
nsiv
e an
tige
n-bi
ndin
g re
pert
oire
, a fi
ndin
g th
at c
alls
into
que
stio
n th
e ro
le o
f lig
ht c
hain
s in
the
cam
el. C
amel
hea
vy-c
hain
IgG
s la
ck C
H1,
whi
ch in
one
IgG
cla
ss m
ight
be
stru
ctur
ally
rep
lace
d by
an
ext
ende
d hi
nge.
Hea
vy-c
hain
IgG
s ar
e a
feat
ure
of a
ll ca
mel
ids.
The
se fi
ndin
gs o
pen
new
per
spec
tive
s in
the
engi
neer
ing
of a
ntib
odie
s.
Ham
er-
scas
ter-
man
et
al. 1
993
Nur
se sh
ark
New
ant
igen
rec
epto
rSe
quen
ce a
naly
sis
Rea
rran
gem
ent
Som
atic
div
ersi
ficat
ion
Imm
unog
lobu
lin a
nd T
-cel
l rec
epto
r (T
CR)
mol
ecul
es a
re c
entr
al to
the
adap
tive
imm
une
syst
em, S
eque
nce
cons
erva
tion
, si
mila
riti
es in
dom
ain
stru
ctur
e, a
nd u
sage
of s
imila
r re
com
bina
tion
sig
nal s
eque
nces
and
rec
ombi
nati
on m
achi
nery
indi
cate
th
at th
ere
was
pro
babl
y a
tim
e du
ring
evo
luti
on w
hen
an a
nces
tral
rec
epto
r di
verg
ed to
the
mod
ern-
day
imm
unog
lobu
lin
and
TC
R(1–
3). O
ther
mol
ecul
es th
at u
nder
go r
earr
ange
men
t hav
e no
t bee
n de
scri
bed
in v
erte
brat
es, n
or h
ave
inte
rmed
iate
s be
en id
enti
fied
that
hav
e fe
atur
es o
f bot
h th
ese
gene
fam
ilies
, We
repo
rt h
ere
the
isol
atio
n of
a n
ew m
embe
r of
the
imm
uno-
glob
ulin
sup
erfa
mily
from
the
nurs
e sh
ark,
Gin
glym
osto
ma
cirr
atum
, whi
ch c
onta
ins
one
vari
able
and
five
con
stan
t dom
ains
an
d is
foun
d as
a d
imer
in s
erum
. Ana
lyse
s of
com
plem
enta
ry D
NA
clo
nes
show
ext
ensi
ve s
eque
nce
dive
rsit
y w
ithin
var
iabl
e do
mai
ns, w
hich
is g
ener
ated
by
both
rea
rran
gem
ent a
nd s
omat
ic d
iver
sific
atio
n m
echa
nism
s. O
ur r
esul
ts s
ugge
st th
at r
ear-
rang
ing
loci
dis
tinc
t fro
m im
mun
oglo
bulin
and
TC
R ha
ve a
rise
n du
ring
evo
luti
on.
Gre
en-
berg
et
al. 1
995
Am
ino
acid
sub
stit
utio
nD
isul
phid
e bo
ndC
yste
in
The
ant
igen
-bin
ding
frag
men
t of f
unct
iona
l hea
vy c
hain
ant
ibod
ies
(HC
Abs
) in
cam
elid
s co
mpr
ises
a s
ingl
e do
mai
n, n
amed
th
e va
riab
le d
omai
n of
hea
vy c
hain
of H
CA
bs (V
HH
). T
he V
HH
har
bors
rem
arka
ble
amin
o ac
id s
ubst
ituti
ons
in th
e fr
ame-
wor
k re
gion
-2 to
gen
erat
e an
ant
igen
-bin
ding
dom
ain
that
func
tion
s in
the
abse
nce
of a
ligh
t cha
in p
artn
er. T
he s
ubst
ituti
ons
prov
ide
a m
ore
hydr
ophi
lic, h
ence
mor
e so
lubl
e, c
hara
cter
to th
e V
HH
but
dec
reas
e th
e in
trin
sic
stab
ility
of t
he d
omai
n.
Her
e w
e in
vest
igat
e th
e fu
ncti
onal
rol
e of
an
addi
tion
al h
allm
ark
of d
rom
edar
y V
HH
s, i.
e. th
e ex
tra
disu
lfide
bon
d be
twee
n th
e fir
st a
nd th
ird
anti
gen-
bind
ing
loop
s. A
fter
sub
stitu
ting
the
cyst
eine
s fo
rmin
g th
is in
terl
oop
cyst
ine
by a
ll 20
am
ino
acid
s, w
e se
lect
ed a
nd c
hara
cter
ized
sev
eral
VH
Hs
that
ret
ain
anti
gen
bind
ing
capa
city
. Alth
ough
VH
H d
omai
ns c
an fu
nc-
tion
in th
e ab
senc
e of
an
inte
rloo
p di
sulfi
de b
ond,
we
dem
onst
rate
that
its
pres
ence
con
stitu
tes
a ne
t adv
anta
ge. F
irst
, the
di
sulfi
de b
ond
stab
ilize
s th
e do
mai
n an
d co
unte
ract
s th
e de
stab
iliza
tion
by
the
fram
ewor
k re
gion
-2 h
allm
ark
amin
o ac
ids.
Se
cond
, the
dis
ulfid
e bo
nd r
igid
ifies
the
long
thir
d an
tige
n-bi
ndin
g lo
op, l
eadi
ng to
a s
tron
ger
anti
gen
inte
ract
ion.
Thi
s du
al
bene
ficia
l eff
ect e
xpla
ins
the
in v
ivo
anti
body
mat
urat
ion
proc
ess
favo
ring
VH
H d
omai
ns w
ith a
n in
terl
oop
disu
lfide
bon
d.
Gov
aert
et
al.
2012
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
453
Am
ino
acid
alig
nmen
tC
DR
iden
tific
atio
nC
onfo
rmat
ion
stud
y
Cam
elid
s hav
e a
spec
ial t
ype
of A
b, k
now
n as
hea
vy c
hain
Abs
, whi
ch a
re d
evoi
d of
cla
ssic
al A
b lig
ht c
hain
s. R
elat
ive
to c
lass
ical
A
bs, c
amel
id h
eavy
cha
in A
bs (c
Abs
) hav
e co
mpa
rabl
e im
mun
ogen
icity
, Ag
reco
gniti
on d
iver
sity
and
bin
ding
affi
nitie
s, h
ighe
r sta
-bi
lity
and
solu
bilit
y, an
d be
tter
man
ufac
tura
bilit
y, m
akin
g th
em p
rom
isin
g ca
ndid
ates
for a
ltern
ate
ther
apeu
tic sc
affol
ds. R
atio
nal
engi
neer
ing
of c
Abs
to im
prov
e th
erap
eutic
func
tion
requ
ires
kno
wle
dgeo
f the
diff
eren
ces o
f seq
uenc
e an
d st
ruct
ural
feat
ures
be
twee
n cA
bs a
nd c
lass
ical
Abs
. In
this
stud
y, am
ino
acid
sequ
ence
s of 2
7 cA
b va
riab
le re
gion
s (V
(H)H
) wer
e al
igne
d w
ith th
e re
spec
tive
regi
ons o
f 54
clas
sica
l Abs
to d
etec
t am
ino
acid
diff
eren
ces,
ena
blin
g au
tom
atic
iden
tifica
tion
of c
Ab
V(H
)H C
DRs
. CD
R an
alys
is re
veal
ed th
at th
e H
1 of
ten
(and
som
etim
es th
e H
2) a
dopt
s div
erse
con
form
atio
ns n
ot c
lass
ifiab
le b
y es
tabl
ishe
d ca
noni
cal
rule
s. A
lso,
alth
ough
the
cAb
H3
is m
uch
long
er th
an c
lass
ical
H3
loop
s, it
oft
en c
onta
ins c
omm
on st
ruct
ural
mot
ifs a
nd so
me-
times
a d
isul
fide
bond
to th
e H
1. L
ever
agin
g th
ese
obse
rvat
ions
, we
crea
ted
a M
onte
Car
lo-b
ased
cA
b V
(H)H
stru
ctur
al m
odel
ing
tool
, whe
re th
e C
DR
H1
and
H2
loop
s exh
ibite
d a
med
ian
root
-mea
n-sq
uare
dev
iatio
n to
nat
ives
of 3
.1 a
nd 1
.5 a
ngst
rom
, res
pec-
tivel
y. Th
e pr
otoc
ol g
ener
ated
8–1
2, 1
4–16
, and
16–
24 re
sidu
e H
3 lo
ops w
ith a
med
ian
root
-mea
n-sq
uare
dev
iatio
n to
nat
ives
of
5.7,
4.5
, and
6.8
ang
stro
m, r
espe
ctiv
ely.
The
larg
e de
viat
ion
of th
e pr
edic
ted
loop
s und
ersc
ores
the
chal
leng
e in
mod
elin
g su
ch lo
ng
loop
s. c
Ab
V(H
)H h
omol
ogy
mod
els c
an p
rovi
de st
ruct
ural
insi
ghts
into
inte
ract
ion
mec
hani
sms t
o en
able
dev
elop
men
t of n
ovel
A
bs fo
r the
rape
utic
and
bio
tech
nolo
gica
l use
.
Sirc
ar e
t al
. 201
1
Prim
ary
stru
ctur
eM
ALD
I-T
OF/
TO
FLC
-MS/
MS
Sequ
enci
ng
The
prim
ary
stru
ctur
e of
a 1
3.6
kDa
sing
le h
eavy
cha
in c
amel
id a
ntib
ody
(V(H
)H) w
as d
eter
min
ed b
y m
atri
x-as
sist
ed la
ser d
esor
p-tio
n io
niza
tion-
time-
of-fl
ight
/tim
e-of
-flig
ht (M
ALD
I-TO
F/TO
F) to
p-do
wn
sequ
ence
ana
lysi
s. Th
e m
ajor
ity o
f the
sequ
ence
was
ob
tain
ed b
y m
ass s
pect
rom
etri
c de
nov
o se
quen
cing
, with
the
N-t
erm
inal
14
amin
o ac
id re
sidu
es b
eing
det
erm
ined
usi
ng T
(3)-
sequ
enci
ng a
nd d
atab
ase
inte
rrog
atio
n. Th
e de
term
ined
sequ
ence
was
con
firm
ed b
y liq
uid
chro
mat
ogra
phy
tand
em m
ass s
pec-
trom
etry
(LC
-MS/
MS)
ana
lysi
s of a
tryp
tic d
iges
t, w
hich
als
o pr
ovid
ed h
igh-
ener
gy c
ollis
iona
lly in
duce
d di
ssoc
iatio
n (C
ID) d
ata
perm
ittin
g th
e cl
ear a
ssig
nmen
t of t
hree
of t
he 1
4 is
obar
ic L
eu/I
le re
sidu
es. F
ive
of th
e 11
Leu
/Ile
am
bigu
ities
cou
ld b
e re
solv
ed b
y ho
mol
ogy
com
pari
sons
with
kno
wn
V(H
)H se
quen
ces.
The
mon
oiso
topi
c m
olec
ular
wei
ght o
f the
was
det
erm
ined
by
ultr
ahig
h-re
solu
tion
orth
ogon
al e
lect
rosp
ray
(ESI
)-TO
F an
alys
is a
nd fo
und
to b
e 13
610
.606
6 D
a, in
exc
elle
nt a
gree
men
t with
the
esta
blis
hed
sequ
ence
. To
our k
now
ledg
e, th
is is
the
first
tim
e th
at th
e en
tire
prim
ary
stru
ctur
e of
a p
rote
in w
ith a
mol
ecul
ar w
eigh
t > 1
3 kD
a ha
s bee
n es
tabl
ishe
d by
mas
s spe
ctro
met
ric
top-
dow
n se
quen
cing
.
Rese
-m
ann
et
al. 2
010
VH
H/h
apte
n
inte
ract
ions
Azo
-dye
X-r
ay s
truc
ture
Cam
elid
s, c
amel
s an
d lla
mas
, hav
e a
uniq
ue im
mun
e sy
stem
abl
e to
pro
duce
hea
vy-c
hain
onl
y an
tibo
dies
. The
ir V
H d
omai
ns
(VH
Hs)
are
the
smal
lest
bin
ding
uni
ts p
rodu
ced
by im
mun
e sy
stem
s, a
nd th
eref
ore
suit
able
for
biot
echn
olog
ical
app
licat
ions
th
roug
h he
tero
logo
us e
xpre
ssio
n. T
he r
ecog
niti
on o
f pro
tein
ant
igen
s by
thes
e V
HH
s is
rat
her
wel
l doc
umen
ted,
whi
le le
ss
is k
now
n ab
out t
he V
HH
/hap
ten
inte
ract
ions
. The
rec
ently
rep
orte
d X
-ray
str
uctu
re o
f a V
HH
in c
ompl
ex w
ith a
cop
per-
cont
aini
ng a
zo-d
ye s
ettle
d th
e ab
ility
of V
HH
to r
ecog
nize
hap
tens
by
form
ing
a ca
vity
bet
wee
n th
e th
ree
com
plem
enta
rity
-de
term
inin
g re
gion
s (C
DR)
. Her
e w
e re
port
the
stru
ctur
es o
f a V
HH
(VH
H A
52) f
ree
or c
ompl
exed
with
an
azo-
dye,
RR1
, w
ithou
t met
al io
n. T
he s
truc
ture
of t
he c
ompl
ex il
lust
rate
s th
e in
volv
emen
t of C
DR2
, CD
R3 a
nd a
fram
ewor
k re
sidu
e in
a
late
ral i
nter
acti
on w
ith th
e ha
pten
. Suc
h a
late
ral c
ombi
ning
site
is c
ompa
rabl
e to
that
foun
d in
cla
ssic
al a
ntib
odie
s, a
lthou
gh
in th
e ab
senc
e of
the
VL.
Spin
elli
et a
l. 20
01
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
454
Caff
eine
Met
hylx
anth
ines
Hea
t-st
abili
tyEL
ISA
We
have
isol
ated
and
cha
ract
eriz
ed a
caff
eine
-spe
cific
, hea
vy-c
hain
-onl
y an
tibod
y fr
agm
ent (
V-H
H) f
rom
llam
a th
at is
cap
able
of
bein
g ut
ilize
d to
ana
lyze
caff
eine
in h
ot a
nd c
old
beve
rage
s. C
amel
id sp
ecie
s (lla
ma
and
cam
el) w
ere
sele
cted
for i
mm
uniz
atio
n be
caus
e of
thei
r pot
entia
l to
mak
e he
at-s
tabl
e, h
eavy
-cha
in-o
nly
antib
odie
s. L
lam
as a
nd c
amel
s wer
e im
mun
ized
with
caff
eine
co
vale
ntly
link
ed to
key
hole
lim
pet h
emoc
yani
n, a
nd re
com
bina
nt a
ntib
ody
tech
niqu
es w
ere
used
to c
reat
e ph
age
disp
laye
d lib
rar-
ies o
f var
iabl
e re
gion
frag
men
ts o
f the
hea
vy-c
hain
ant
ibod
ies.
Caff
eine
-spe
cific
VH
H fr
agm
ents
wer
e se
lect
ed b
y th
eir a
bilit
y to
bi
nd to
caff
eine
/bov
ine
seru
m a
lbum
in (B
SA) a
nd c
onfir
med
by
a po
sitiv
e re
actio
n in
a c
affei
ne e
nzym
e-lin
ked
imm
unos
orbe
nt
assa
y (c
affei
ne E
LISA
). O
ne o
f the
se V
HH
frag
men
ts (V
SA2)
was
exp
ress
ed a
s a so
lubl
e pr
otei
n an
d sh
own
to re
cove
r its
reac
tiv-
ity a
fter
exp
osur
e to
tem
pera
ture
s up
to 9
0 de
gree
s C. I
n ad
ditio
n, V
SA2
was
abl
e to
bin
d ca
ffein
e at
70
degr
ees C
. A c
ompe
titio
n ca
ffein
e EL
ISA
was
dev
elop
ed fo
r the
mea
sure
men
t of c
affei
ne in
bev
erag
es, a
nd c
once
ntra
tions
of c
affei
ne o
btai
ned
for c
offee
, C
oca-
Col
a C
lass
ic, a
nd D
iet C
oke
agre
ed w
ell w
ith h
igh
perf
orm
ance
liqu
id c
hrom
atog
raph
y (H
PLC
) det
erm
inat
ion
and
liter
atur
e va
lues
. VSA
2 sh
owed
min
imal
cro
ss re
activ
ity w
ith st
ruct
ural
ly re
late
d m
ethy
lxan
thin
es.
Lade
n-so
n et
al.
2006
Met
hotr
exat
eIm
mun
e-lla
ma
libra
ryA
nti-
MT
X V
HH
gen
esH
ydro
solu
ble
hapt
en
Sing
le-d
omai
n an
tibod
ies s
peci
fic to
met
hotr
exat
e (M
TX
) wer
e ob
tain
ed a
fter
imm
uniz
atio
n of
one
llam
a (L
lam
a gl
ama)
. Spe
cific
V
HH
dom
ains
(V-D
-J-r
egio
n) w
ere
sele
cted
by
pann
ing
from
an
imm
une-
llam
a lib
rary
usi
ng p
hage
dis
play
tech
nolo
gy. Th
e an
ti-bo
dy fr
agm
ents
spec
ific
to M
TX
wer
e pu
rifie
d fr
om E
sche
rich
ia c
oli (
C41
stra
in) p
erip
lasm
by
imm
obili
zed
met
al a
ffini
ty c
hrom
a-to
grap
hy w
ith a
n ex
pres
sion
leve
l of a
roun
d 10
mg/
l. A
sing
le b
and
arou
nd 1
6 00
0 D
a co
rres
pond
ing
to V
HH
frag
men
ts w
as fo
und
afte
r ana
lysi
s by
SDS-
PAG
E an
d W
este
rn b
lott
ing,
whi
le c
ompe
titio
n EL
ISA
dem
onst
rate
d se
lect
ive
bind
ing
to so
lubl
e M
TX
. Su
rfac
e pl
asm
on re
sona
nce
(SPR
) ana
lysi
s sho
wed
that
ant
i-MT
X V
HH
dom
ains
had
affi
nitie
s in
the
nano
mol
ar ra
nge
(29–
515
nM) t
o M
TX
-ser
um a
lbum
in c
onju
gate
s. Th
e ge
nes e
ncod
ing
anti-
MT
X V
HH
wer
e fo
und
by IM
GT
/V-Q
UES
T to
be
sim
ilar t
o th
e pr
evio
usly
repo
rted
llam
a an
d hu
man
IGH
V g
erm
line
gene
s. Th
e V
-D a
nd D
-J ju
nctio
n re
arra
ngem
ents
in th
e se
ven
anti-
MT
X
CD
R3 se
quen
ces i
ndic
ate
that
they
wer
e or
igin
ated
from
thre
e di
stin
ct p
roge
nito
r B c
ells
. Our
resu
lts d
emon
stra
te th
at c
amel
id
sing
le-d
omai
n an
tibod
ies a
re c
apab
le o
f hig
h affi
nity
bin
ding
to lo
w m
olec
ular
wei
ght h
ydro
solu
ble
hapt
ens.
Fur
ther
mor
e, th
ese
anti-
MT
X V
HH
giv
e ne
w in
sigh
ts o
n ho
w th
e an
tigen
bin
ding
repe
rtoi
re o
f lla
ma
sing
le-d
omai
n an
tibod
y ca
n pr
ovid
e co
mbi
ning
si
tes t
o ha
pten
s in
the
abse
nce
of a
VL.
This
type
of s
ingl
e-do
mai
n an
tibod
ies o
ffers
adv
anta
ges c
ompa
red
to m
urin
e re
com
bina
nt
antib
odie
s in
term
s of p
rodu
ctio
n ra
te a
nd se
quen
ce si
mila
rity
to th
e hu
man
IGH
V3
subg
roup
gen
es.
Alv
arez
-Ru
eda
et
al. 2
007
C(H
)1 d
omai
n ab
senc
eSe
quen
ce a
naly
sis
Splic
ing
The
mol
ecul
ar b
asis
for t
he a
bsen
ce o
f the
C(H
)1 d
omai
n in
nat
ural
ly o
ccur
ring
hea
vy-c
hain
ant
ibod
ies o
f the
cam
elid
s was
ass
esse
d by
det
erm
inin
g th
e en
tire
Cam
elus
dro
med
ariu
s gam
ma
2a h
eavy
-cha
in c
onst
ant g
ene.
The
orga
niza
tion
of th
e ca
mel
gam
ma
2a c
on-
stan
t hea
vy-c
hain
gen
e ob
tain
ed fr
om a
live
r gen
omic
libr
ary
appe
ars t
o be
typi
cal o
f all
othe
r mam
mal
ian
gam
ma
gene
s seq
uenc
ed to
da
te. I
t con
tain
s the
switc
h, C
(H)1
, hin
ge, C
(H)2
, C(H
)3, M
1 an
d M
2 ex
ons.
In c
ontr
ast t
o th
e ca
se in
mou
se a
nd h
uman
hea
vy c
hain
di
seas
es, t
he c
amel
gam
ma
2a g
ene
show
s no
maj
or st
ruct
ural
def
ect,
and
its e
quiv
alen
t C(H
)1 e
xon
is in
tact
. How
ever
, seq
uenc
e an
alys
is h
as re
veal
ed th
at th
e sp
licin
g si
te, i
mm
edia
tely
afte
r the
C(H
)1 e
xon,
is d
efec
tive
due
to p
oint
mut
atio
ns, e
spec
ially
the
G(+
1) to
A(+
1) tr
ansv
ersi
on se
ems t
o be
det
rim
enta
l. It
is c
oncl
uded
that
the
loss
of t
he sp
lice
cons
ensu
s sig
nal i
s res
pons
ible
for t
he
rem
oval
of t
he e
ntire
C(H
)1 d
omai
n in
cam
el g
amm
a 2a
hea
vy-c
hain
imm
unog
lobu
lins.
Add
ition
ally
, a c
lose
r ana
lysi
s of t
he h
inge
ex
on su
gges
ts th
e po
ssib
le in
volv
emen
t of t
rans
poso
ns in
the
gene
tic v
aria
tion
of m
amm
alia
n C
gam
ma
hing
es.
Ngu
yen
et a
l. 19
99
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
455
Inhi
bito
ry a
ntib
odie
sEn
zym
e’s
acti
ve s
ite
Car
boni
c an
hydr
ase
Alp
ha-a
myl
ase
Rec
ombi
nant
pro
tein
Evid
ence
is p
rovi
ded
that
dro
med
ary
heav
y-ch
ain
antib
odie
s, in
viv
o-m
atur
ed in
the
abse
nce
of li
ght c
hain
s, a
re a
uni
que
sour
ce o
f inh
ibito
ry a
ntib
odie
s. A
fter
imm
uniz
atio
n of
a d
rom
edar
y w
ith b
ovin
e er
ythr
ocyt
e ca
rbon
ic a
nhyd
rase
and
por
cine
pa
ncre
atic
alp
ha-a
myl
ase,
it w
as d
emon
stra
ted
that
a c
onsi
dera
ble
amou
nt o
f hea
vy-c
hain
ant
ibod
ies,
act
ing
as tr
ue c
ompe
titiv
e in
hibi
tors
, cir
cula
te in
the
bloo
dstr
eam
. In
cont
rast
, the
con
vent
iona
l ant
ibod
ies
appa
rent
ly d
o no
t int
erac
t with
the
enzy
me’s
ac
tive
site
. Nex
t we
illus
trat
ed th
at p
erip
hera
l blo
od ly
mph
ocyt
es a
re s
uita
ble
for
one-
step
clo
ning
of t
he v
aria
ble
dom
ain
frag
-m
ents
in a
pha
ge-d
ispl
ay v
ecto
r. By
bio
-pan
ning
, sev
eral
ant
igen
-spe
cific
sin
gle-
dom
ain
frag
men
ts a
re re
adily
isol
ated
for
both
en
zym
es. I
n ad
ditio
n w
e sh
ow th
at a
mon
g th
ose
isol
ated
frag
men
ts a
ctiv
e si
te b
inde
rs a
re w
ell r
epre
sent
ed. W
hen
prod
uced
as
reco
mbi
nant
pro
tein
in E
sche
rich
ia c
oli,
thes
e ac
tive
site
bin
ders
app
ear
to b
e po
tent
enz
yme
inhi
bito
rs w
hen
test
ed in
chr
o-m
ogen
ic a
ssay
s. T
he lo
w c
ompl
exity
of t
he a
ntig
en-b
indi
ng s
ite o
f the
se s
ingl
e-do
mai
n an
tibod
ies
com
pose
d of
onl
y th
ree
loop
s co
uld
be v
alua
ble
for
desi
gnin
g sm
alle
r sy
nthe
tic in
hibi
tors
.
Lauw
-er
eys e
t al
. 199
8
VH
HA
ntig
en-b
indi
ng lo
ops
RN
ase
AEn
zym
e in
hibi
tor
Can
onic
al s
truc
ture
s
Back
grou
nd: C
amel
id s
erum
con
tain
s a
larg
e fr
actio
n of
func
tiona
l hea
vy c
hain
ant
ibod
ies
– ho
mod
imer
s of
hea
vy c
hain
s w
ithou
t lig
ht c
hain
s. T
he v
aria
ble
dom
ains
of t
hese
hea
vy-c
hain
ant
ibod
ies
(VH
H) h
ave
a lo
ng c
ompl
emen
tari
ty d
eter
min
ing
regi
on 3
(CD
R3) l
oop
that
com
pens
ates
for
the
abse
nce
of th
e an
tigen
-bin
ding
loop
s of
the
vari
able
ligh
t cha
ins
(VL)
. In
the
case
of t
he V
HH
frag
men
t cA
b-Ly
s3, p
art o
f the
24
amin
o ac
id lo
ng C
DR3
loop
pro
trud
es fr
om th
e an
tigen
-bin
ding
sur
face
an
d in
sert
s in
to th
e ac
tive-
site
cle
ft o
f its
ant
igen
, ren
deri
ng c
Ab-
Lys3
a c
ompe
titiv
e en
zym
e in
hibi
tor.
Resu
lts: A
dro
med
ary
VH
H w
ith s
peci
ficity
for
bovi
ne R
Nas
e A
, cA
b-RN
05, h
as a
sho
rt C
DR3
loop
of 1
2 am
ino
acid
s an
d is
not
a c
ompe
titiv
e en
zym
e in
hibi
tor.
The
str
uctu
re o
f the
cA
b-RN
05-R
Nas
e A
com
plex
has
bee
n so
lved
at 2
.8 a
ngst
rom
. The
VH
H s
caffo
ld a
rchi
tect
ure
is c
lose
to th
at o
f a h
uman
VH
(var
iabl
e he
avy
chai
n). T
he s
truc
ture
of t
he a
ntig
en-b
indi
ng h
yper
vari
able
1 lo
op (H
1) o
f bot
h cA
b-RN
05 a
nd c
Ab-
Lys3
diff
er fr
om th
e kn
own
cano
nica
l str
uctu
res;
in a
dditi
on th
ese
H1
loop
s re
sem
ble
each
oth
er. T
he
CD
R3 p
rovi
des
an a
ntig
en-b
indi
ng s
urfa
ce a
nd s
hiel
ds th
e fa
ce o
f the
dom
ain
that
inte
ract
s w
ith V
L in
con
vent
iona
l ant
ibod
ies.
C
oncl
usio
ns: V
HH
s ad
opt t
he c
omm
on im
mun
oglo
bulin
fold
of v
aria
ble
dom
ains
, but
the
antig
en-b
indi
ng lo
ops
devi
ate
from
th
e pr
edic
ted
cano
nica
l str
uctu
re. W
e de
fine
a ne
w c
anon
ical
str
uctu
re fo
r th
e H
1 lo
op o
f im
mun
oglo
bulin
s, w
ith c
Ab-
RN05
an
d cA
b-Ly
s3 a
s re
fere
nce
stru
ctur
es. T
his
new
loop
str
uctu
re m
ight
als
o oc
cur
in h
uman
or
mou
se V
H d
omai
ns. S
urpr
isin
gly,
only
two
loop
s ar
e in
volv
ed in
ant
igen
reco
gniti
on; t
he C
DR2
doe
s no
t par
ticip
ate.
Nev
erth
eles
s, th
e an
tigen
bin
ding
occ
urs
with
nan
omol
ar a
ffin
ities
bec
ause
of a
pre
fere
ntia
l usa
ge o
f mai
ncha
in a
tom
s fo
r an
tigen
inte
ract
ion.
Dec
an-
nier
e et
al
. 199
9
Cle
fts
on p
rote
in
surf
aces
C
onve
x pa
rato
peA
ntig
en-c
ombi
ning
sit
eLy
sozy
me
inhi
bito
rC
ryst
al s
truc
ture
Cle
fts
on p
rote
in s
urfa
ces
are
avoi
ded
by a
ntig
en-c
ombi
ning
site
s of
con
vent
iona
l ant
ibod
ies,
in c
ontr
ast t
o he
avy-
chai
n an
tibod
ies
(HC
Abs
) of c
amel
ids
that
see
m to
be
attr
acte
d by
enz
ymes
’ sub
stra
te p
ocke
ts. T
he e
xpla
natio
n fo
r th
is p
rono
unce
d pr
efer
ence
of H
CA
bs w
as in
vest
igat
ed. E
ight
sin
gle
dom
ain
antig
en-b
indi
ng fr
agm
ents
of H
CA
bs (V
HH
) with
nan
omol
ar a
ffin
i-tie
s fo
r ly
sozy
me
wer
e is
olat
ed fr
om th
ree
imm
uniz
ed d
rom
edar
ies.
Six
of e
ight
VH
Hs
com
pete
with
sm
all l
ysoz
yme
inhi
bito
rs.
Thi
s ra
tio o
f act
ive
site
bin
ders
is a
lso
foun
d w
ithin
the
VH
H p
ool d
eriv
ed fr
om p
olyc
lona
l HC
Abs
pur
ified
from
the
seru
m o
f th
e im
mun
ized
dro
med
ary.
The
cry
stal
str
uctu
res
of s
ix V
HH
s in
com
plex
with
lyso
zym
e an
d th
eir
inte
ract
ion
surf
aces
wer
e co
mpa
red
to th
ose
of c
onve
ntio
nal a
ntib
odie
s w
ith th
e sa
me
antig
en. T
he in
terf
ace
size
s of
VH
H a
nd c
onve
ntio
nal a
ntib
odie
s to
lyso
zym
e ar
e ve
ry s
imila
r as
wel
l as
the
num
ber
and
chem
ical
nat
ure
of th
e co
ntac
ts. T
he m
ain
diffe
renc
e co
mes
from
the
com
pact
pro
late
sha
pe o
f VH
H th
at p
rese
nts
a la
rge
conv
ex p
arat
ope,
pre
dom
inan
tly fo
rmed
by
the
H3
loop
and
inte
ract
ing,
al
thou
gh w
ith d
iffer
ent s
truc
ture
s, in
to th
e co
ncav
e ly
sozy
me
subs
trat
e-bi
ndin
g po
cket
. The
refo
re, a
sin
gle
dom
ain
antig
en-
com
bini
ng s
ite h
as a
cle
ar s
truc
tura
l adv
anta
ge o
ver
a co
nven
tiona
l dim
eric
form
at fo
r ta
rget
ing
clef
ts o
n an
tigen
ic s
urfa
ces.
De
Gen
st
et a
l. 20
06
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
456
Car
tila
gino
us fi
shcD
NA
seq
uenc
e an
alys
isIg
NA
RIg
W
The
cart
ilagi
nous
fish
(chi
mer
as, s
hark
s, sk
ates
and
rays
) are
the
olde
st g
roup
rela
tive
to m
amm
als i
n w
hich
an
adap
tive
imm
une
syst
em fo
unde
d up
on im
mun
oglo
bulin
s has
bee
n fo
und.
In th
is m
anus
crip
t we
char
acte
rize
the
imm
unog
lobu
lins o
f the
spin
y do
gfish
(S
qual
us a
cant
hias
) at b
oth
the
mol
ecul
ar a
nd e
xpre
ssed
pro
tein
leve
ls. D
espi
te th
e pr
esen
ce o
f hun
dred
s of I
gM c
lust
ers i
n th
is
spec
ies t
he se
rum
leve
ls of
this
isot
ype
are
com
para
tivel
y lo
w. H
owev
er, a
naly
sis o
f cD
NA
sequ
ence
s and
seru
m p
rote
in su
gges
ts
mic
rohe
tero
gene
ity in
the
IgM
hea
vy c
hain
s and
supp
orts
the
prop
osal
that
diff
eren
t clu
ster
s are
pre
fere
ntia
lly u
sed
in th
e tw
o fo
rms
(mon
omer
or p
enta
mer
) of t
his i
soty
pe. W
e al
so fo
und
that
the
IgN
AR
isot
ype
in th
is sp
ecie
s exi
sts i
n a
prev
ious
ly u
nkno
wn
mul
ti-m
eric
form
at in
seru
m. F
inal
ly, w
e id
entifi
ed a
new
form
of t
he Ig
W is
otyp
e (th
e sh
ark
IgD
ort
holo
gue)
, in
whi
ch th
e le
ader
is sp
liced
di
rect
ly to
the
first
con
stan
t dom
ain,
resu
lting
in a
mol
ecul
e la
ckin
g an
ant
igen
-bin
ding
dom
ain.
Smith
et
al. 2
012
V(N
AR
) dom
ain
Rib
osom
e di
spla
yEr
ror-
pron
e
mut
agen
esis
Mut
atio
nal p
last
icit
ySt
ruct
ural
mod
ellin
g
The
shar
k an
tigen
-bin
ding
V(N
AR)
dom
ain
has t
he p
oten
tial t
o pr
ovid
e an
att
ract
ive
alte
rnat
ive
to tr
aditi
onal
bio
ther
apeu
tics
base
d on
its s
mal
l siz
e, a
dvan
tage
ous p
hysi
oche
mic
al p
rope
rtie
s, a
nd u
nusu
al a
bilit
y to
targ
et c
left
s in
enzy
mes
or c
ell s
urfa
ce
mol
ecul
es. Th
e V
(NA
R) sh
ares
man
y of
the
prop
ertie
s of t
he w
ell-c
hara
cter
ised
sing
le-d
omai
n ca
mel
id V
(H)H
but
is m
uch
less
un
ders
tood
at t
he m
olec
ular
leve
l. W
e ch
ose
the
hen-
egg-
lyso
zym
e-sp
ecifi
c ar
chet
ypal
Typ
e I V
(NA
R) 5
A7
and
used
ribo
som
e di
spla
y in
com
bina
tion
with
err
or-p
rone
mut
agen
esis
to in
terr
ogat
e th
e en
tire
sequ
ence
spac
e. W
e fo
und
a hi
gh le
vel o
f mut
atio
nal
plas
ticity
acr
oss t
he V
(NA
R) d
omai
n, p
artic
ular
ly w
ithin
the
fram
ewor
k 2
and
hype
rvar
iabl
e re
gion
2 re
gion
s. A
num
ber o
f res
i-du
es im
port
ant f
or a
ffini
ty w
ere
iden
tified
, and
a tr
iple
mut
ant c
ombi
ning
AID
, S61
R, a
nd G
62R
resu
lted
in a
K(D
) of 4
60 p
M fo
r he
n eg
g ly
sozy
me,
a 2
0-fo
ld im
prov
emen
t ove
r wild
-typ
e 5A
7, a
nd th
e hi
ghes
t K(D
) yet
repo
rted
for V
(NA
R)-a
ntig
en in
tera
ctio
ns.
Thes
e fin
ding
s wer
e ra
tiona
lised
usi
ng st
ruct
ural
mod
ellin
g an
d in
dica
te th
e im
port
ance
of r
esid
ues o
utsi
de th
e cl
assi
cal c
ompl
e-m
enta
rity
det
erm
inin
g re
gion
s in
mak
ing
nove
l ant
igen
con
tact
s tha
t mod
ulat
e affi
nity
. We
also
loca
ted
two
solv
ent-
expo
sed
resi
-du
es (G
15 a
nd G
42),
dist
ant f
rom
the
V(N
AR)
par
atop
e, w
hich
reta
in fu
nctio
n up
on m
utat
ion
to c
yste
ine
and
have
the
pote
ntia
l to
be e
xplo
ited
as si
tes f
or ta
rget
ed c
oval
ent m
odifi
catio
n. O
ur fi
ndin
gs w
ith 5
A7
wer
e ex
tend
ed to
all
know
n N
AR
stru
ctur
es u
sing
an
in-d
epth
bio
info
rmat
ic a
naly
sis o
f seq
uenc
e da
ta a
vaila
ble
in th
e lit
erat
ure
and
a ne
wly
gen
erat
ed V
(NA
R) d
atab
ase.
This
stud
y al
low
ed u
s to
iden
tify,
for t
he fi
rst t
ime,
bot
h V
(NA
R)-s
peci
fic a
nd V
(NA
R)/I
g V
(L)/
TCR
V(a
lpha
) ove
rlap
ping
hal
lmar
k re
sidu
es,
whi
ch a
re c
ritic
al fo
r the
stru
ctur
al a
nd fu
nctio
nal i
nteg
rity
of t
he si
ngle
dom
ain.
Intr
igui
ngly
, eac
h of
our
des
igna
ted
V(N
AR)
-sp
ecifi
c ha
llmar
ks a
lign
prec
isel
y w
ith p
revi
ousl
y de
fined
mut
atio
nal ‘
cold
spot
s’ in
nat
ural
nur
se sh
ark
cDN
A se
quen
ces.
Thes
e fin
ding
s will
aid
futu
re V
(NA
R) e
ngin
eeri
ng a
nd o
ptim
isat
ion
stud
ies t
owar
ds th
e de
velo
pmen
t of V
(NA
R) si
ngle
-dom
ain
prot
eins
as
via
ble
biot
hera
peut
ics.
Fenn
ell
et a
l. 20
10
IgM
IgN
AR
IgW
Hou
ndsh
ark
In th
is st
udy,
cDN
As e
ncod
ing
the
secr
eted
form
s of t
he im
mun
oglo
bulin
(Ig)
hea
vy c
hain
s of I
gM, I
gNA
R, a
nd Ig
W w
ere
clon
ed
from
the
band
ed h
ound
shar
k Tr
iaki
s scy
llium
. Tw
o cl
ones
for t
he Ig
M h
eavy
cha
ins e
ncod
ed 5
69 a
nd 5
70 a
min
o ac
ids,
who
se c
on-
serv
ed (C
) reg
ion
show
ed 4
7–70
% a
min
o ac
id id
entit
ies t
o th
ose
repo
rted
in o
ther
car
tilag
inou
s fish
. Fou
r clo
nes f
or th
e Ig
NA
R en
code
d 67
3–67
0 am
ino
acid
s with
con
serv
ed Ig
-sup
erfa
mily
dom
ains
. The
IgN
AR
C re
gion
show
ed 5
6–69
% a
min
o ac
id id
enti-
ties t
o th
ose
so fa
r rep
orte
d. H
igh-
thro
ughp
ut se
quen
cing
reve
aled
that
in m
ost o
f the
IgN
AR
sequ
ence
s, th
e tw
o va
riab
le re
gion
s (C
DR1
and
CD
R3) e
ach
poss
ess a
cys
tein
e re
sidu
e. Th
ree
type
s of I
gW w
ere
iden
tified
; one
con
tain
ed Ig
-sup
erfa
mily
dom
ains
th
at a
re in
oth
er c
artil
agin
ous fi
sh, o
ne la
cks t
he 3
rd d
omai
n in
the
cons
tant
regi
on, a
nd o
ne la
cks t
he 3
rd to
5th
dom
ains
. Des
pite
th
ese
diffe
renc
es, t
he Ig
W is
ofor
ms c
lust
ered
with
IgW
s of o
ther
car
tilag
inou
s fish
es a
nd th
e C
regi
ons s
how
ed 4
7–89
% a
min
o ac
id
iden
titie
s. m
RNA
s for
IgM
and
IgN
AR
wer
e de
tect
ed in
var
ious
tiss
ues,
whi
le Ig
W m
RNA
was
mai
nly
dete
cted
in p
ancr
eas.
The
band
ed h
ound
ed sh
ark
also
has
IgM
, IgW
and
IgN
AR
as w
ell a
s the
oth
er c
artil
agin
ous fi
sh w
ith u
niqu
e Ig
W is
ofor
m.
Hon
da e
t al
. 201
0
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
457
New
ant
igen
rec
epto
rD
isul
phid
e bo
ndLo
op s
tabi
lizat
ion
Cry
stal
logr
aphi
c st
udie
s
The
new
ant
igen
rece
ptor
(IgN
AR)
ant
ibod
ies f
rom
shar
ks a
re d
isul
phid
e bo
nded
dim
ers o
f tw
o pr
otei
n ch
ains
, eac
h co
ntai
ning
on
e va
riab
le a
nd fi
ve c
onst
ant d
omai
ns. Th
ree
type
s of I
gNA
R va
riab
le d
omai
ns h
ave
been
dis
cove
red,
with
Typ
e 3
appe
arin
g ea
rly
in sh
ark
deve
lopm
ent a
nd b
eing
ove
rtak
en b
y th
e an
tigen
-dri
ven
affini
ty-m
atur
ed T
ype
1 an
d 2
resp
onse
. Her
e, w
e ha
ve d
eter
-m
ined
the
first
stru
ctur
e of
a n
atur
ally
occ
urri
ng T
ype
2 Ig
NA
R va
riab
le d
omai
n, a
nd id
entifi
ed th
e di
sulp
hide
bon
d th
at li
nks a
nd
stab
ilize
s the
CD
R1 a
nd C
DR3
loop
s. Th
is d
isul
phid
e br
idge
lock
s the
CD
R3 lo
op in
an
“upr
ight
” con
form
atio
n in
con
tras
t to
othe
r sh
ark
antib
ody
stru
ctur
es, w
here
a m
ore
late
ral c
onfig
urat
ion
is o
bser
ved.
Fur
ther
, we
soug
ht to
mod
el th
e Ty
pe 3
isot
ype
base
d on
th
e cr
ysta
llogr
aphi
c st
ruct
ure
repo
rted
her
e. Th
is m
odel
incr
indi
cate
s tha
t int
erna
l Typ
e 3-
spec
ific
resi
dues
com
bine
to p
ack
into
a
com
pact
imm
unog
lobu
lin c
ore
that
supp
orts
the
CD
R lo
op re
gion
s, a
nd th
at d
espi
te a
ppar
ent l
ow-s
eque
nce
vari
abili
ty, t
here
Is
suffi
cien
t pla
stic
ity in
the
CD
R3 lo
op to
form
a c
onfo
rmat
iona
lly d
iver
se a
ntig
en-b
indi
ng su
rfac
e.
Stre
ltsov
et
al.
2005
Ant
ibod
y ev
olut
ion
Phyl
ogen
etic
and
phe
no-
typi
c re
lati
onsh
ips
Cam
elid
aeSh
arks
The
emer
genc
e in
Cam
elid
ae sp
ecie
s of f
unct
iona
l ant
ibod
ies d
evoi
d of
ligh
t cha
ins (
refe
rred
to a
s hea
vy-c
hain
ant
ibod
ies o
r H
CA
bs) i
s an
intr
igui
ng e
volu
tiona
ry e
vent
. Hom
odim
eric
HC
Abs
hav
e al
so b
een
docu
men
ted
in sp
otte
d ra
tfish
(Cos
5-A
bs) a
nd
nurs
e sh
ark
(NA
R). T
o re
veal
the
evol
utio
nary
his
tory
of H
CA
bs, w
e ev
alua
ted
the
phyl
ogen
etic
and
phe
noty
pic
rela
tions
hips
am
ong
HC
Abs
and
con
vent
iona
l ant
ibod
ies a
cros
s tax
a an
d co
nfirm
ed th
e cu
rren
t vie
wpo
int t
hat d
iffer
ent g
roup
s of H
CA
bs h
ave
evol
ved
inde
pend
ently
in th
e th
ree
linea
ges.
At l
east
, in
the
cam
elid
s, H
CA
bs a
re n
ot th
e re
sult
of re
susc
itatio
n of
dor
man
t gen
es.
They
are
der
ived
from
the
conv
entio
nal a
ntib
odie
s with
in th
e C
amel
idae
line
age,
and
are
app
aren
tly th
e ou
tcom
e of
mor
e re
cent
ad
aptiv
e ch
ange
s occ
urri
ng in
the
com
part
men
t of h
eter
omer
ic a
ntib
odie
s. Th
e sh
ared
stru
ctur
al p
rope
rtie
s of H
CA
bs a
cros
s tax
a ar
e th
eref
ore
expl
aine
d by
con
verg
ent e
volu
tion
due
to si
mila
r con
stra
ints
rela
ted
to th
e ab
senc
e of
pai
ring
to th
e lig
ht c
hain
. It
appe
ars t
hat i
nnov
ativ
e ev
olut
iona
ry c
hang
es in
Cam
elid
ae h
ave
led
to a
new
leve
l of a
ntig
en b
indi
ng re
pert
oire
div
ersi
ficat
ion
and
have
allo
wed
acq
uisi
tion
of n
ovel
ant
igen
-rec
epto
r pro
pert
ies.
Ngu
yen
et a
l. 20
02
Ada
ptiv
e im
mun
e re
spon
seM
icro
bial
enz
yme
in
hibi
tion
P-la
ctam
ase
In 1
993,
a fr
actio
n of
ant
ibod
ies (
Abs
) dev
oid
of L
cha
in w
as fo
und
natu
rally
occ
urri
ng in
the
Cam
elid
ae. Th
ey w
ere
foun
d to
lack
L
chai
ns, a
s wel
l as t
he fi
rst c
onst
ant h
eavy
-cha
in d
omai
n (C
HI)
and
ther
efor
e th
ey w
ere
nam
ed “h
eavy
-cha
in A
bs” (
HC
Abs
). Su
bseq
uent
stud
ies f
ocus
ed o
n th
e fu
nctio
nal,
stru
ctur
al a
nd b
ioch
emic
al p
rope
rtie
s of r
ecom
bina
nt v
aria
ble
frag
men
ts (r
VH
Hs)
of
HC
Abs
. It w
as st
ated
that
rVH
Hs h
ave
an a
ugm
ente
d ca
paci
ty to
inte
ract
with
“par
tially
hid
den”
epi
tope
s, li
ke e
nzym
es a
ctiv
e si
tes,
and
hav
e an
incr
ease
d st
abili
ty to
ther
mal
and
che
mic
al a
ggre
ssio
n. It
has
bee
n su
gges
ted
that
thes
e un
conv
entio
nal A
bs
coul
d re
pres
ent a
n ev
olut
iona
ry a
dvan
tage
, bei
ng m
ore
effici
ent t
han
conv
entio
nal A
bs to
inhi
bit m
icro
bial
enz
ymes
, and
thus
ex
ertin
g a
mor
e pr
otec
tive
imm
une
resp
onse
aga
inst
pat
hoge
ns. Th
e pr
esen
t wor
k fo
cuse
s on
the
imm
unob
iolo
gica
l rol
e of
H
CA
bs, i
n th
eir c
apac
ity to
inhi
bit m
icro
bial
enz
ymes
. Tw
o an
imal
mod
els w
ere
sele
cted
, com
pris
ing
a m
odel
for c
omm
on v
er-
tebr
ates
with
out H
CA
bs (r
abbi
ts),
and
a m
odel
for v
erte
brat
es w
ith b
oth
conv
entio
nal a
nd u
ncon
vent
iona
l Abs
(Lam
a gl
ama)
. A
reco
mbi
nant
bac
teri
al P
-lact
amas
e (C
TX
-M-2
) was
sele
cted
as t
he m
icro
bial
enz
ymat
ic a
ntig
en. A
fter
con
vent
iona
l im
mun
izat
ion
sche
dule
s, n
eith
er se
rum
tite
rs n
or se
rum
inhi
bito
ry c
apac
ity sh
owed
sign
ifica
nt d
iffer
ence
s whe
n ra
bbits
and
llam
as w
ere
com
-pa
red.
Thes
e re
sults
indi
cate
that
the
a pr
iori
ass
umpt
ion
that
the
adap
tive
imm
une
syst
em o
f cam
elid
s cou
ld b
e be
tter
“pre
pare
d”
to re
spon
d to
bac
teri
al e
nzym
es b
ecau
se o
f the
pre
senc
e of
HC
Abs
, is n
ot a
lway
s acc
urat
e. F
urth
erm
ore,
whe
n th
e di
ffere
nt ll
ama
antib
ody
isot
ypes
and
subc
lass
es w
ere
puri
fied,
it w
as d
emon
stra
ted
that
the
inhi
bito
ry c
apac
ity o
f tot
al se
rum
was
due
exc
lusi
vely
to
IgG
I. H
CA
bs n
ot o
nly
faile
d to
inhi
bit C
TX
-M-2
, but
inst
ead
they
act
ivat
ed it
s enz
ymat
ic a
ctiv
ity. A
ltoge
ther
, the
se re
sults
in
dica
te th
at th
e hy
poth
eses
ext
rapo
late
d fr
om th
e rV
HH
s pro
pert
ies n
eed
to b
e re
vise
d; th
e re
al ro
le o
f HC
Abs
in v
ivo
rem
ains
un
know
n, a
s wel
l as t
heir
evo
lutio
nary
cau
se.
Ferr
ari e
t al
. 200
7
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
458
Imm
unol
ogic
al
prop
erti
esC
ompl
emen
t act
ivat
ing
acti
vity
Ant
icom
plem
enta
ry
acti
vity
Hem
aggl
utin
atio
n
Hea
vy c
hain
ant
ibod
ies (
HC
Abs
), de
void
of t
he li
ght c
hain
s and
the
CH
1 do
mai
n, a
re p
rese
nt in
the
seru
m o
f cam
elid
s. Ig
G(2
) an
d Ig
G(3
) are
HC
Abs
; whe
reas
IgG
(1) h
as th
e co
nven
tiona
l str
uctu
re. I
n or
der t
o st
udy
the
imm
unol
ogic
al p
rope
rtie
s of l
lam
a H
CA
bs, f
rom
whi
ch to
dat
e lit
tle is
kno
wn,
llam
as (L
ama
glam
a) H
CA
bs c
DN
A w
ere
clon
ed, s
eque
nced
and
com
pare
d w
ith o
ther
m
amm
alia
n Ig
s. Th
e se
quen
ce a
naly
sis s
how
ed th
at ll
ama
HC
Abs
cD
NA
org
aniz
atio
n is
sim
ilar t
o ot
her m
amm
alia
n Ig
s and
the
pres
ence
of c
onse
rved
bin
ding
mot
ifs to
Pro
tein
A, P
rote
in G
, Fc
gam
ma
RI, F
c ga
mm
a RI
II a
nd C
1q in
HC
Abs
wer
e ob
serv
ed. I
n a
prev
ious
wor
k, d
iffer
ent I
gG is
otyp
es p
urifi
ed b
y Pr
otei
n A
and
Pro
tein
G c
hrom
atog
raph
y w
ere
assa
yed
for t
heir
abi
lity
to fi
x co
mpl
emen
t. Bo
th Ig
G(1
) and
the
tota
l ser
um w
ere
able
to fi
x co
mpl
emen
t, w
here
as Ig
G(2
) and
IgG
(3) fi
xed
com
plem
ent e
ven
in
the
abse
nce
of a
ntig
en (a
ntic
ompl
emen
tary
act
ivity
). Th
eref
ore,
in th
is w
ork
we
perf
orm
ed th
e co
mpl
emen
t act
ivat
ing
activ
ity o
f th
e di
ffere
nt Ig
G is
otyp
es p
urifi
ed u
nder
phy
siol
ogic
al c
ondi
tions
usi
ng S
epha
dex
G-1
50 a
nd th
eir a
bilit
y to
indu
ce h
emag
glut
ina-
tion.
Lla
mas
wer
e im
mun
ized
with
shee
p re
d bl
ood
cells
(RBC
) str
oma
and
the
diffe
rent
isot
ypes
wer
e pu
rifie
d fr
om se
ra. W
hole
se
rum
and
IgG
(1) c
ould
act
ivat
e co
mpl
emen
t; ho
wev
er, H
CA
bs (I
gG(2
) + Ig
G(3
)) c
ould
not
, des
pite
the
pres
ence
of t
he C
1q b
ind-
ing
mot
if in
thei
r pri
mar
y se
quen
ce. U
nlik
e Ig
G(1
), th
e fr
actio
n co
rres
pond
ing
to Ig
G(2
) + Ig
G(3
) did
not
dis
play
hem
aggl
utin
atin
g ac
tivity
. Our
find
ings
sugg
est t
hat H
CA
bs c
anno
t cro
sslin
k effi
cien
tly w
ith d
iffer
ent a
ntig
ens a
nd th
at th
e C
1q b
indi
ng si
te m
ight
be
hin
dere
d by
the
prox
imity
of t
he v
aria
ble
dom
ains
.
Sacc
o-do
ssi e
t al
. 201
2
Ant
igen
-bin
ding
re
pert
oire
Ger
mlin
e da
taba
seSe
quen
ce c
ompa
riso
nEv
olut
ion
Som
atic
mut
atio
n
The
antig
en-b
indi
ng si
te o
f the
cam
el h
eavy
-cha
in a
ntib
odie
s dev
oid
of li
ght c
hain
con
sist
s of a
sing
le v
aria
ble
dom
ain
(VH
H)
that
obv
ious
ly la
cks t
he V
-H-V
-L c
ombi
nato
rial
div
ersi
ty, T
o ev
alua
te th
e ex
tent
of t
he V
HH
ant
igen
-bin
ding
repe
rtoi
re, a
ger
-m
line
data
base
was
con
stru
cted
from
PC
R-am
plifi
ed T
HH
/V-H
segm
ents
of a
sing
le sp
ecim
en o
f Cam
elus
dro
med
ariu
s. A
tota
l of
33
VH
H a
nd 3
9 V
H u
niqu
e se
quen
ces w
ere
iden
tified
, enc
oded
by
42 a
nd 5
0 di
ffere
nt g
enes
, res
pect
ivel
y. Se
quen
ce c
ompa
ri-
son
indi
cate
s tha
t the
V(H
)Hs e
volv
ed w
ithin
the
VH
subg
roup
III,
Nev
erth
eles
s, th
e V
HH
ger
mlin
e se
gmen
ts a
re h
ighl
y di
vers
e,
lead
ing
to a
bro
ad st
ruct
ural
repe
rtoi
re o
f the
ant
igen
-bin
ding
loop
s, S
even
VH
H su
bfam
ilies
wer
e re
cogn
ized
, of w
hich
five
wer
e co
nfirm
ed to
be
expr
esse
d lit
viv
o. C
ompa
riso
n of
ger
mlin
e an
d cD
NA
sequ
ence
s dem
onst
rate
s tha
t the
rear
rang
ed V
(H)H
s are
ex
tens
ivel
y di
vers
ified
by
som
atic
mut
atio
n pr
oces
ses,
lead
ing
to a
n ad
ditio
nal h
yper
vari
able
regi
on a
nd a
hig
h in
cide
nce
of n
ucle
-ot
ide
inse
rtio
ns o
r del
etio
ns, Th
ese
dive
rsifi
catio
n pr
oces
ses a
re d
rive
n by
hyp
erm
utat
ion
and
reco
mbi
natio
n ho
tspo
ts e
mbe
dded
in
the
VH
H g
erm
line
gene
s at t
he re
gion
s affe
ctin
g th
e st
ruct
ure
of th
e an
tigen
-bin
ding
loop
s.
Ngu
yen
et a
l. 20
00
App
licat
ion
Hum
an le
ukoc
yte
an
tige
nC
ance
r
A n
ovel
mon
oclo
nal a
nti-
pan
hum
an le
ukoc
yte
antig
en (H
LA) c
lass
I he
avy
chai
n an
tibod
y, EM
R8-5
, was
est
ablis
hed.
It c
ould
de
tect
HLA
-A, -
B, a
nd -C
ant
igen
s in
form
alin
-fixe
d pa
raffi
n em
bedd
ed ti
ssue
s. B
y im
mun
ohis
toch
emic
al st
aini
ng u
sing
the
EMR8
-5 a
ntib
ody,
vari
ous c
ance
r tis
sues
from
246
cas
es w
ere
exam
ined
for H
LA c
lass
I ex
pres
sion
. It w
as fo
und
that
HLA
cla
ss I
expr
essi
on w
as d
ecre
ased
in 2
0% to
42%
of t
he c
ases
of l
ung
canc
er, h
epat
ocel
lula
r car
cino
ma,
col
on c
ance
r, re
nal c
ell c
arci
nom
a,
and
urot
helia
l car
cino
ma.
In c
ontr
ast,
85%
of b
reas
t can
cer c
ases
had
loss
of o
r dec
reas
ed H
LA c
lass
I ex
pres
sion
. Of t
he 3
5 br
east
ca
ncer
cas
es th
at h
ad d
ecre
ased
HLA
cla
ss I
heav
y ch
ain
expr
essi
on, 3
3 (9
4%) a
lso
had
decr
ease
d be
ta2-
mic
rogl
obul
in e
xpre
ssio
n de
tect
ed b
y im
mun
ohis
toch
emic
al st
aini
ng. I
t was
sugg
este
d th
at H
LA c
lass
I do
wn-
regu
latio
n m
ight
be
a co
mm
on c
hara
cter
istic
of
bre
ast c
ance
r mos
tly c
ause
d by
the
dow
n-re
gula
tion
of b
eta2
-mic
rogl
obul
in e
xpre
ssio
n.
Tori
goe
et a
l. 20
12
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
459
App
licat
ion
Prio
n im
mun
othe
rapy
Scra
pie
PrP
conf
orm
atio
n
Alth
ough
ther
e is
cur
rent
ly n
o eff
ectiv
e tr
eatm
ent f
or p
rion
dis
ease
s, si
gnifi
cant
adv
ance
s hav
e be
en m
ade
in su
ppre
ssin
g its
pr
ogre
ss, u
sing
ant
ibod
ies t
hat b
lock
the
conv
ersi
on o
f PrP
(C) i
nto
PrP(
Sc).
In o
rder
to b
e eff
ectiv
e in
trea
ting
indi
vidu
als t
hat
have
pri
on d
isea
ses,
ant
ibod
ies m
ust b
e ca
pabl
e of
arr
estin
g di
seas
e in
its l
ate
stag
es. Th
is re
quir
es th
e de
velo
pmen
t of a
ntib
od-
ies w
ith h
ighe
r affi
nity
for P
rP(S
c) a
nd sy
stem
s for
effe
ctiv
e tr
ansl
ocat
ion
of a
ntib
odie
s acr
oss t
he b
lood
bra
in b
arri
er in
ord
er to
ac
hiev
e hi
gh c
once
ntra
tions
of i
nhib
itor a
t the
site
of p
rote
in re
plic
atio
n. A
n ad
ditio
nal a
dvan
tage
is th
e ab
ility
of t
hese
ant
ibod
ies
to a
cces
s the
cyt
osol
of a
ffect
ed c
ells
. To
this
end
, we
have
gen
erat
ed P
rP-s
peci
fic a
ntib
odie
s (kn
own
as P
rioV
) by
imm
uniz
atio
n of
cam
els w
ith m
urin
e sc
rapi
e m
ater
ial a
dsor
bed
to im
mun
omag
netic
bea
ds. Th
e Pr
ioV
ant
ibod
ies d
ispl
ay a
rang
e of
spec
ifici
ties
with
som
e re
cogn
izin
g th
e Pr
P (2
7–30
) pro
tein
ase
K-re
sist
ant f
ragm
ent,
othe
rs sp
ecifi
c fo
r PrP
(C) a
nd a
num
ber w
ith d
ual b
ind-
ing
spec
ifici
ty. I
ndep
ende
nt o
f the
ir P
rP c
onfo
rmat
ion
spec
ifici
ty, o
ne o
f the
Pri
oV a
ntib
odie
s (Pr
ioV
3) w
as sh
own
to b
ind
PrP(
C)
in th
e cy
toso
l of n
euro
blas
tom
a ce
lls. I
n m
arke
d co
ntra
st, c
onve
ntio
nal a
nti-P
rP a
ntib
odie
s pro
duce
d in
mou
se a
gain
st si
mila
r ta
rget
ant
igen
wer
e un
able
to c
ross
the
neur
onal
pla
sma
mem
bran
e an
d in
stea
d fo
rmed
a ri
ng a
roun
d th
e ce
lls. Th
e Pr
ioV
ant
i-Pr
P an
tibod
ies c
ould
pro
ve to
be
a va
luab
le to
ol fo
r the
neu
tral
izat
ion/
clea
ranc
e of
PrP
(Sc)
in in
trac
ellu
lar c
ompa
rtm
ents
of a
ffect
ed
neur
ons a
nd c
ould
pot
entia
lly h
ave
wid
er a
pplic
abili
ty fo
r the
trea
tmen
t of s
o-ca
lled
prot
ein-
mis
fold
ing
dise
ases
.
Taye
bi e
t al
. 201
0
App
licat
ion
Com
peti
tive
flui
d ar
ray
imm
unoa
ssay
Trin
itro
tolu
ene
Llam
as p
osse
ss u
niqu
e su
bcla
sses
of a
ntib
odie
s tha
t lac
k lig
ht c
hain
s, a
nd th
us a
re m
ade
by th
e pa
irin
g of
two
heav
y ch
ains
. IgG
w
as p
urifi
ed fr
om tw
o lla
mas
whi
ch h
ad b
een
imm
uniz
ed w
ith tr
initr
oben
zene
-key
hole
lim
pet h
emoc
yani
n. C
onve
ntio
nal I
gG1
and
heav
y ch
ain
IgG
2 an
d Ig
G3
subc
lass
es w
ere
frac
tiona
ted
usin
g affi
nity
chr
omat
ogra
phy.
The
effec
tiven
ess o
f hea
vy c
hain
an
tibod
ies f
or th
e de
tect
ion
of tr
initr
otol
uene
(TN
T) u
sing
a c
ompe
titiv
e flu
id a
rray
imm
unoa
ssay
was
eva
luat
ed a
nd c
ompa
red
to
both
the
llam
a Ig
G1
as w
ell a
s a m
urin
e m
onoc
lona
l ant
i-TN
T a
ntib
ody.
It w
as fo
und
that
hea
vy c
hain
ant
ibod
y bo
und
TN
T w
ith
sele
ctiv
ity si
mila
r to
conv
entio
nal a
ntib
odie
s, y
et th
e he
avy
chai
n an
tibod
ies p
osse
ssed
gre
ater
ther
mal
stab
ility
. The
titer
of t
he
heav
y ch
ain
antib
odie
s how
ever
was
foun
d to
be
10-f
old
low
er th
an th
e Ig
Gl1
; thu
s ana
lytic
al a
ssay
s wer
e be
st d
emon
stra
ted
usin
g th
e lla
ma
IgG
1 co
nven
tiona
l ant
ibod
y. Th
e T
NT
com
petit
ive
imm
unoa
ssay
on
the
Lum
inex
flui
d an
alyz
er h
ad a
dyn
amic
rang
e fr
om si
mila
r to
100
ng/m
l to
10 µ
g/m
l. U
tiliz
ing
the
sam
e tw
o-st
ep c
ompe
titiv
e as
say
form
at th
e dy
nam
ic ra
nge
of th
e m
onoc
lona
l an
tibod
y w
as fo
und
to h
ave
a br
oad
rang
e (1
ng/
ml t
o 1
µg/m
l). Th
is m
etho
d w
as d
emon
stra
ted
on T
NT
con
tam
inat
ed so
il ex
trac
ts
usin
g bo
th th
e lla
ma
IgG
1 an
d th
e m
ouse
mon
oclo
nal v
alid
atin
g th
e ut
ility
of m
etho
d fo
r ana
lysi
s of h
eld
sam
ples
.
And
er-
son
and
Gol
dman
20
08
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
460
Tabl
e 4.
Pro
duct
ion
of s
ingl
e-do
mai
n an
tibo
dy fr
agm
ents
usi
ng r
ecom
bina
nt te
chno
logy
E. c
oli
Azo
xyst
robi
nM
etal
affi
nity
chr
oma-
togr
aphy
ELIS
A
Thr
ee V
(H)H
s ag
ains
t the
mod
el h
apte
n, a
zoxy
stro
bin
(MW
403
), w
ere
isol
ated
from
a h
yper
-im
mun
ized
pha
ge-d
ispl
ayed
V
(H)H
libr
ary.
Thi
s lib
rary
was
con
stru
cted
by
isol
atin
g th
e V
(H)H
-cod
ing
gene
s fr
om th
e ly
mph
ocyt
es c
olle
cted
from
a
Lam
a gl
ama
that
was
imm
uniz
ed w
ith a
zoxy
stro
bin
conj
ugat
ed to
bov
ine
seru
m a
lbum
in (B
SA).
Six
roun
ds o
f pan
ning
wer
e pe
rfor
med
aga
inst
azo
xyst
robi
n co
njug
ated
to e
ither
ova
lbum
in (O
VA) o
r ra
bbit
ser
um a
lbum
in (R
SA) t
o en
rich
clo
nes
cont
aini
ng V
(H)H
s sp
ecifi
c to
the
hapt
en. A
fter
scr
eeni
ng 9
5 cl
ones
, thr
ee V
(H)H
s (A
27, A
72, a
nd A
85) w
ith d
iffer
ent a
min
o ac
id s
eque
nces
wer
e id
enti
fied,
exp
ress
ed in
sol
uble
form
at in
Esc
heri
chia
col
i HB2
151,
and
pur
ified
usi
ng n
icke
l-im
mo-
biliz
ed m
etal
aff
init
y ch
rom
atog
raph
y. C
ompe
titi
ve in
hibi
tion
enz
yme-
linke
d im
mun
osor
bent
ass
ay (C
I-EL
ISA
) sho
wed
th
at A
27 a
nd A
85 w
ere
spec
ific
to a
zoxy
stro
bin
whi
le A
72 w
as n
ot. T
he IC
(50)
val
ues
of A
27 a
nd A
85 V
(H)H
s w
ere
7.2
and
2.0
µm, r
espe
ctiv
ely.
To
our
know
ledg
e A
85 is
one
of t
he h
ighe
st a
ffin
ity
V(H
)Hs
that
has
yet
bee
n is
olat
ed a
gain
st a
hyd
ro-
phob
ic h
apte
n su
ch a
s az
oxys
trob
in.
Mak
-va
ndi-
Nej
ad e
t al
. 201
1
Pich
ia p
asto
ris
Aah
I’ sc
orpi
on to
xin
Alc
ohol
oxi
dase
pr
omot
erA
-fac
tor
secr
etio
n si
gnal
Usi
ng e
ukar
yoti
c ho
sts,
ant
ibod
y fr
agm
ents
are
gen
eral
ly e
xpre
ssed
by
targ
etin
g to
the
secr
etor
y pa
thw
ay. T
his
enab
les
not
only
eff
icie
nt d
isul
fide
bond
form
atio
n bu
t als
o se
cret
ion
of s
olub
le a
nd c
orre
ctly
fold
ed p
rodu
ct. F
or th
is g
oal,
a re
com
bi-
nant
vec
tor
was
con
stru
cted
to p
rodu
ce a
sin
gle-
dom
ain
anti
body
(NbA
ahI’2
2) d
irec
ted
agai
nst A
ahI’s
corp
ion
toxi
n us
ing
the
met
hylo
trop
hic
yeas
t. T
he c
orre
spon
ding
com
plem
enta
ry D
NA
was
clo
ned
unde
r co
ntro
l of t
he a
lcoh
ol o
xida
se p
ro-
mot
er in
fram
e w
ith th
e Sa
ccha
rom
yces
α-f
acto
r se
cret
ion
sign
al a
nd th
en tr
ansf
erre
d to
P. p
asto
ris
cell
stra
in X
-33.
Usi
ng
Wes
tern
blo
t, w
e de
tect
ed th
e ex
pres
sion
of t
he r
ecom
bina
nt N
bAah
I’22
excl
usiv
ely
in th
e cu
lture
med
ium
. Tar
geti
ng to
th
e hi
stid
ine
labe
l, th
e se
cret
ed n
anob
ody
was
eas
ily p
urifi
ed o
n ni
ckel
nitr
ilotr
iace
tic
acid
res
in a
nd th
en te
sted
in e
nzym
e-lin
ked
imm
unos
orbe
nt a
ssay
. Int
eres
ting
ly, t
he p
rodu
ctio
n le
vel o
f the
NbA
ahI’2
2 in
its
new
gly
cosy
late
d fo
rm r
each
ed m
ore
than
six
fold
that
obt
aine
d in
E. c
oli.
The
se fi
ndin
gs g
ive
mor
e ev
iden
ce fo
r th
e ut
iliza
tion
of P
. pas
tori
s as
a h
eter
olog
ous
expr
essi
on s
yste
m.
Ezzi
ne
et a
l. 20
12
Sacc
haro
myc
es
cere
visi
ae
Secr
etio
n effi
cien
cyJ s
egm
ent
Vari
able
dom
ains
of l
lam
a he
avy-
chai
n an
tibo
dies
(VH
H) a
re b
ecom
ing
a po
tent
tool
for
a w
ide
rang
e of
bio
tech
nolo
gica
l an
d m
edic
al a
pplic
atio
ns. B
ecau
se o
f str
uctu
ral f
eatu
res
typi
cal o
f the
ir s
ingl
e-do
mai
n na
ture
, the
y ar
e re
lati
vely
eas
y to
pro
-du
ce in
low
er e
ukar
yote
s, b
ut it
is n
ot u
ncom
mon
that
som
e m
olec
ules
hav
e po
or s
ecre
tion
eff
icie
ncy.
We
ther
efor
e se
t out
to
stu
dy th
e pr
oduc
tion
yie
ld o
f VH
H. W
e co
mpu
tati
onal
ly id
enti
fied
five
key
resi
dues
that
are
cru
cial
for
fold
ing
and
secr
e-ti
on, a
nd w
e va
lidat
ed th
eir
impo
rtan
ce w
ith s
yste
mat
ic s
ite-d
irec
ted
mut
atio
ns. T
he o
bser
vati
on th
at a
ll ke
y re
sidu
es w
ere
loca
lised
in th
e V
seg
men
t, in
pro
xim
ity
of th
e J s
egm
ent o
f VH
H, l
ed u
s to
stu
dy th
e im
port
ance
of J
seg
men
t in
secr
etio
n ef
ficie
ncy.
Intr
igui
ngly
, we
foun
d th
at th
e us
e of
spe
cific
J se
gmen
ts in
VH
H c
ould
str
ongl
y in
fluen
ce th
e pr
oduc
tion
yie
ld.
Sequ
ence
ana
lysi
s an
d ex
pres
sion
exp
erim
ents
str
ongl
y su
gges
ted
that
inte
ract
ions
with
cha
pero
nes,
esp
ecia
lly w
ith th
e J
segm
ent,
are
a cr
ucia
l asp
ect o
f the
pro
duct
ion
yiel
d of
VH
H.
Gor
lani
et
al.
2012
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
461
Mag
neto
spir
illum
gr
yphi
swal
dens
e M
agne
tic
nano
part
icle
Mag
neto
som
eR
ed fl
uore
scei
n pr
otei
n
Num
erou
s ap
plic
atio
ns o
f con
vent
iona
l and
bio
geni
c m
agne
tic n
anop
artic
les
(MN
Ps),
such
as
in d
iagn
ostic
s, im
mun
omag
netic
se
para
tions
, and
mag
netic
cel
l lab
elin
g, re
quir
e th
e im
mob
iliza
tion
of a
ntib
odie
s. T
his
is u
sual
ly a
ccom
plis
hed
by c
hem
ical
co
njug
atio
n, w
hich
, how
ever
, has
sev
eral
dis
adva
ntag
es, s
uch
as p
oor
effic
ienc
y an
d th
e ne
ed fo
r co
uplin
g ch
emis
try.
Her
e,
we
desc
ribe
a n
ovel
str
ateg
y to
dis
play
a fu
nctio
nal c
amel
id a
ntib
ody
frag
men
t (na
nobo
dy) f
rom
an
alpa
ca (L
ama
paco
s) o
n th
e su
rfac
e of
bac
teri
al b
ioge
nic
mag
netic
nan
opar
ticle
s (m
agne
toso
mes
). M
agne
toso
me-
spec
ific
expr
essi
on o
f a re
d flu
ores
-ce
nt p
rote
in (R
FP)-
bind
ing
nano
body
(RBP
) in
vivo
was
acc
ompl
ishe
d by
gen
etic
fusi
on o
f RBP
to th
e m
agne
toso
me
prot
ein
Mam
C in
the
mag
netit
e-sy
nthe
sizi
ng b
acte
rium
Mag
neto
spir
illum
gry
phis
wal
dens
e. W
e de
mon
stra
te th
at is
olat
ed m
agne
to-
som
es e
xpre
ssin
g M
amC
-RBP
eff
icie
ntly
reco
gniz
e an
d bi
nd th
eir
antig
en in
vitr
o an
d ca
n be
use
d fo
r im
mun
opre
cipi
tatio
n of
RFP
-tag
ged
prot
eins
and
thei
r in
tera
ctio
n pa
rtne
rs fr
om c
ell e
xtra
cts.
In a
dditi
on, w
e sh
ow th
at c
oexp
ress
ion
of m
onom
eric
RF
P (m
RFP
or it
s va
rian
t mC
herr
y) a
nd M
amC
-RBP
resu
lts in
intr
acel
lula
r re
cogn
ition
and
mag
neto
som
e re
crui
tmen
t of R
FP
with
in li
ving
bac
teri
a. T
he in
trac
ellu
lar
expr
essi
on o
f a fu
nctio
nal n
anob
ody
targ
eted
to a
spe
cific
bac
teri
al c
ompa
rtm
ent o
pens
ne
w p
ossi
bilit
ies
for
in v
ivo
synt
hesi
s of
MN
P-im
mob
ilize
d na
nobo
dies
. Mor
eove
r, in
trac
ellu
lar
nano
trap
s ca
n be
gen
erat
ed to
m
anip
ulat
e ba
cter
ial s
truc
ture
s in
live
cel
ls.
Polli
thy
et a
l. 20
11
Toba
cco
expr
essi
on
syst
emA
grob
acte
rium
Seve
ral p
lant
s are
the
faci
le a
nd e
cono
mic
bio
reac
tor f
or la
rge-
scal
e pr
oduc
tion
of in
dust
rial
and
pha
rmac
eutic
al a
gent
s lik
e pr
o-te
ins a
nd a
ntib
odie
s. H
ere,
we
have
sele
cted
toba
cco
as th
e ho
st p
lant
bec
ause
of l
arge
scal
e pr
oduc
tion
capa
bilit
y an
d m
any
othe
r ad
vant
ages
such
as g
reat
er sa
fety
and
low
er p
rodu
ctio
n co
sts w
hen
com
pare
d to
ani
mal
-bas
ed sy
stem
s. In
this
stud
y, w
e ha
ve su
b-cl
oned
VH
H g
ene
into
pBI
121
usin
g ph
asm
id p
CA
NTA
B5E.
The
new
con
stru
ct w
as u
sed
to tr
ansf
orm
the
Agr
obac
teri
um st
rain
s C
58G
V31
01 a
nd L
BA44
04. A
grob
acte
rium
stra
in C
58G
V31
01 sh
owed
a h
ighe
r vir
ulen
ce o
n le
af d
isks
of N
icot
iana
taba
cum
(N
C25
). Tr
ansg
enic
toba
cco
plan
ts w
ere
then
dev
elop
ed b
y in
trod
ucin
g V
HH
gen
e un
der t
he c
ontr
ol o
f CaM
V 3
5S p
rom
oter
. The
pres
ence
of t
he V
HH
ant
ibod
y ge
ne in
the
plan
t gen
ome
was
ver
ified
by
PCR
anal
ysis
and
Sou
ther
n hy
brid
izat
ion
expe
rim
ents
. N
orth
ern
blot
ana
lysi
s sho
wed
that
the
gene
s cod
ing
for t
he V
HH
cou
ld b
e ex
pres
sed
in to
bacc
o pl
ants
. Thre
e lin
es o
f tra
nsge
nic
plan
t tha
t exp
ress
es h
igh
leve
ls o
f mRN
A w
ere
scre
ened
in a
furt
her a
naly
sis.
The
expr
essi
on o
f VH
H w
as th
en o
bser
ved
in tr
ans-
geni
c pl
ants
by
ELIS
A u
sing
the
spec
ific
antib
ody
deve
lope
d, th
e re
sults
show
ed th
ree
to fi
ve fo
lds h
ighe
r tha
n no
n-tr
ansg
enic
to
bacc
os.
Kor
ou-
zhde
hy
et a
l. 20
11
Tum
our
necr
osis
fact
orFu
sion
Elas
tin-
like
poly
pept
ide
Toba
cco
Tum
our n
ecro
sis f
acto
r (T
NF)
is a
maj
or p
ro-in
flam
mat
ory
cyto
kine
invo
lved
in m
ultip
le in
flam
mat
ory
dise
ases
. The
detr
imen
tal
activ
ity o
f TN
F ca
n be
blo
cked
by
vari
ous a
ntag
onis
ts, a
nd c
omm
erci
al th
erap
eutic
s bas
ed u
pon
this
pri
ncip
le h
ave
been
app
rove
d fo
r tre
atm
ent o
f dis
ease
s inc
ludi
ng rh
eum
atoi
d ar
thri
tis, C
rohn
’s di
seas
e an
d ps
oria
sis.
In a
sear
ch fo
r new
, im
prov
ed a
nti-
infla
mm
ator
y th
erap
eutic
s we
have
des
igne
d a
sing
le-d
omai
n m
onoc
lona
l ant
ibod
y (V
(H)H
), w
hich
reco
gniz
es T
NF.
The
antib
ody
com
pone
nt (T
NF-
V(H
)H) i
s bas
ed u
pon
an a
nti-h
uman
TN
F C
amel
idae
hea
vy-c
hain
mon
oclo
nal a
ntib
ody,
whi
ch w
as li
nked
to a
n el
astin
-like
pol
ypep
tide
(ELP
). W
e de
mon
stra
te th
at E
LP fu
sion
to th
e T
NF-
V(H
)H e
nhan
ces a
ccum
ulat
ion
of th
e fu
sion
pro
tein
du
ring
bio
man
ufac
turi
ng in
tran
sgen
ic to
bacc
o pl
ants
. With
this
stud
y, w
e sh
ow fo
r the
firs
t tim
e th
at th
is p
lant
-der
ived
ant
i-hu
man
TN
F-V
(H)H
ant
ibod
y w
as b
iolo
gica
lly a
ctiv
e in
viv
o. Th
eref
ore,
ther
apeu
tic a
pplic
atio
n of
TN
F-V
(H)H
-ELP
fusi
on p
rote
in
was
test
ed in
hum
aniz
ed T
NF
mic
e an
d w
as sh
own
to b
e eff
ectiv
e in
pre
vent
ing
deat
h ca
used
by
sept
ic sh
ock.
The
in v
ivo
pers
is-
tenc
e of
the
ELPy
late
d an
tibod
y w
as si
mila
r to
24 fo
ld lo
nger
than
that
of n
on-E
LPyl
ated
TN
F-V
(H)H
.
Con
rad
et a
l. 20
11
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
462
Pann
ing
met
hods
Bot
ulin
um n
euro
toxi
nB
indi
ng k
inet
ics
Sing
le-d
omai
n an
tibo
dies
(sdA
b) s
peci
fic fo
r bo
tulin
um n
euro
toxi
n se
roty
pe A
(BoN
T A
) wer
e se
lect
ed fr
om a
n im
mun
e lla
ma
phag
e di
spla
y lib
rary
der
ived
from
a ll
ama
that
was
imm
uniz
ed w
ith B
oNT
A to
xoid
. The
con
stru
cted
pha
ge li
brar
y w
as p
anne
d us
ing
two
met
hods
: pan
ning
on
plat
es c
oate
d w
ith B
oNT
A to
xoid
(BoN
T A
Td) a
nd B
oNT
A c
ompl
ex to
xoid
(B
oNT
Ac
Td) a
nd p
anni
ng o
n m
icro
sphe
res
coup
led
to B
oNT
A T
d an
d Bo
NT
A to
xin
(BoN
T A
Tx)
. Bot
h pa
nnin
g m
etho
ds
sele
cted
for
bind
ers
that
had
iden
tica
l seq
uenc
es, s
ugge
stin
g th
at p
anni
ng o
n to
xoid
ed m
ater
ial m
ay b
e as
eff
ecti
ve a
s pa
n-ni
ng o
n be
ad-i
mm
obili
zed
toxi
n fo
r is
olat
ing
spec
ific
bind
ers.
All
of th
e is
olat
ed b
inde
rs te
sted
wer
e ob
serv
ed to
rec
ogni
ze
bead
-im
mob
ilize
d Bo
NT
A T
x in
dir
ect b
indi
ng a
ssay
s, a
nd s
how
ed v
ery
little
cro
ss-r
eact
ivit
y to
war
ds o
ther
BoN
T s
ero-
type
s an
d un
rela
ted
prot
ein.
San
dwic
h as
says
that
inco
rpor
ated
sel
ecte
d sd
Ab
as c
aptu
re a
nd tr
acer
ele
men
ts d
emon
stra
ted
that
all
of th
e sd
Ab
wer
e ab
le to
rec
ogni
ze s
olub
le (“
live”
) BoN
T A
Tx
and
BoN
T A
c Tx
with
vir
tual
ly n
o cr
oss-
reac
tivi
ty
with
oth
er B
oNT
ser
otyp
es. T
he is
olat
ed s
dAb
did
not e
xhib
it th
e hi
gh d
egre
e of
ther
mal
sta
bilit
y of
ten
asso
ciat
ed w
ith
thes
e re
agen
ts; a
fter
the
first
hea
ting
cyc
le m
ost o
f the
bin
ding
act
ivit
y w
as lo
st, b
ut th
e po
rtio
n of
the
prot
ein
that
did
ref
old
and
reco
ver
anti
gen-
bind
ing
acti
vity
sho
wed
onl
y m
inim
al lo
ss o
n su
bseq
uent
hea
ting
and
coo
ling
cycl
es. T
he b
indi
ng k
inet
-ic
s of
sel
ecte
d bi
nder
s, a
sses
sed
by b
oth
an e
quili
briu
m fl
uid
arra
y as
say
as w
ell a
s su
rfac
e pl
asm
on r
eson
ance
(SPR
) usi
ng
toxo
ided
mat
eria
l, ga
ve d
isso
ciat
ion
cons
tant
s (K
(D))
in th
e ra
nge
2.2
× 10
(–11
) to
1.6
× 10
(–10
)M. T
hese
hig
h-af
finit
y bi
nd-
ers
may
pro
ve b
enef
icia
l to
the
deve
lopm
ent o
f rec
ombi
nant
rea
gent
s fo
r th
e ra
pid
dete
ctio
n of
BoN
T A
, par
ticu
larl
y in
fiel
d sc
reen
ing
and
mon
itori
ng a
pplic
atio
ns.
Swai
n et
al
. 201
0
Picl
oram
Rib
osom
e di
spla
yIn
vit
ro tr
ansc
ript
ion/
tr
ansl
atio
nPo
int m
utat
ion
Bin
ding
kin
etic
sM
olec
ular
evo
luti
on
Picl
oram
-spe
cific
var
iabl
e fr
agm
ents
(V(H
H)s
) of h
eavy
cha
in a
ntib
odie
s (H
CA
bs) w
ere
sele
cted
from
a n
aive
-llam
a lib
rary
usi
ng
ribo
som
e di
spla
y te
chno
logy
. A c
DN
A li
brar
y of
V(H
H)s
was
con
stru
cted
from
lym
phoc
ytes
of a
non
-im
mun
ized
llam
a an
d en
gi-
neer
ed to
allo
w in
vitr
o tr
ansc
ript
ion
and
tran
slat
ion.
With
no
stop
cod
ons p
rese
nt o
n th
e tr
ansc
ript
s, tr
imer
ic c
ompl
exes
of r
ibo-
som
es, m
RNA
s and
nas
cent
pep
tides
wer
e pr
oduc
ed fo
r affi
nity
sele
ctio
n, i.
e. p
anni
ng. A
fter
thre
e cy
cles
of p
anni
ng, s
even
diff
er-
ent V
(HH
)s a
ll be
long
ing
to th
e V
-HH
subf
amily
1 w
ere
isol
ated
. Fol
low
ing
anot
her t
hree
cyc
les o
f sel
ectio
n, o
nly
two
of th
e se
ven
V(H
H)s
per
sist
ed. A
com
pari
son
of th
ese
two
sequ
ence
s with
kno
wn
sequ
ence
s in
the
liter
atur
e su
gges
ts th
at p
oint
mut
atio
ns
may
hav
e be
en in
trod
uced
into
the
DN
A p
ool d
urin
g PC
R am
plifi
catio
n st
eps o
f lib
rary
con
stru
ctio
n, p
anni
ng a
nd/o
r clo
ning
. Th
ree
sepa
rate
poi
nt m
utat
ions
cau
sing
thre
e in
depe
nden
t am
ino
acid
cha
nges
(non
syno
nom
ous m
utat
ions
) acc
umul
ated
in th
e sa
me
sequ
ence
and
enr
iche
d th
roug
hout
the
sele
ctio
n pr
otoc
ol, s
ugge
stin
g th
at th
ese
chan
ges c
onfe
r bin
ding
adv
anta
ges.
Sur
face
pl
asm
on re
sona
nce
(SPR
) ana
lysi
s was
use
d to
det
erm
ine
bind
ing
kine
tics o
f the
two
clon
es (3
-1D
2 an
d 3-
1F6)
repr
esen
ting
the
two
diffe
rent
sets
of i
sola
ted
com
plem
enta
rity
det
erm
inin
g re
gion
(CD
R)3s
. Mea
sure
d K
(D)s
wer
e 3
and
254
µM, r
espe
ctiv
ely.
The
resu
lts in
dica
te th
at ri
boso
me
disp
lay
tech
nolo
gy c
an b
e us
ed to
effi
cien
tly is
olat
e ha
pten
-spe
cific
ant
ibod
y (A
b) fr
agm
ents
from
a
naiv
e lib
rary
and
con
curr
ently
intr
oduc
e di
vers
ity to
the
sele
cted
poo
l the
reby
faci
litat
ing
mol
ecul
ar e
volu
tion.
Rib
osom
e di
spla
y te
chno
logy
can
com
pens
ate
for t
he li
mite
d di
vers
ity o
f a V
-HH
nai
ve li
brar
y an
d pr
ovid
e an
unl
imite
d so
urce
of a
ffini
ty-m
atur
ed
imm
unoa
ctiv
e re
agen
ts in
vitr
o.
Yau
et
al. 2
003
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
463
Rib
osom
e di
spla
yEr
ror-
pron
e
mut
agen
esis
Lyso
zym
eSo
lven
t-ex
pose
d
resi
dues
We
chos
e th
e he
n-eg
g-ly
sozy
me-
spec
ific
arch
etyp
al T
ype
I V(N
AR)
5A
7 an
d us
ed ri
boso
me
disp
lay
in c
ombi
natio
n w
ith e
rror
-pr
one
mut
agen
esis
to in
terr
ogat
e th
e en
tire
sequ
ence
spac
e. W
e fo
und
a hi
gh le
vel o
f mut
atio
nal p
last
icity
acr
oss t
he V
(NA
R)
dom
ain,
par
ticul
arly
with
in th
e fr
amew
ork
2 an
d hy
perv
aria
ble
regi
on 2
regi
ons.
A n
umbe
r of r
esid
ues i
mpo
rtan
t for
affi
n-ity
wer
e id
entifi
ed, a
nd a
trip
le m
utan
t com
bini
ng A
ID, S
61R
, and
G62
R re
sulte
d in
a K
(D) o
f 460
pM
for h
en e
gg ly
sozy
me,
a
20-f
old
impr
ovem
ent o
ver w
ild-t
ype
5A7,
and
the
high
est K
(D) y
et re
port
ed fo
r V(N
AR)
-ant
igen
inte
ract
ions
. Thes
e fin
ding
s wer
e ra
tiona
lised
usi
ng st
ruct
ural
mod
ellin
g an
d in
dica
te th
e im
port
ance
of r
esid
ues o
utsi
de th
e cl
assi
cal c
ompl
emen
tari
ty d
eter
min
-in
g re
gion
s in
mak
ing
nove
l ant
igen
con
tact
s tha
t mod
ulat
e affi
nity
. We
also
loca
ted
two
solv
ent-
expo
sed
resi
dues
(G15
and
G42
), di
stan
t fro
m th
e V
(NA
R) p
arat
ope,
whi
ch re
tain
func
tion
upon
mut
atio
n to
cys
tein
e an
d ha
ve th
e po
tent
ial t
o be
exp
loite
d as
si
tes f
or ta
rget
ed c
oval
ent m
odifi
catio
n. O
ur fi
ndin
gs w
ith 5
A7
wer
e ex
tend
ed to
all
know
n N
AR
stru
ctur
es u
sing
an
in-d
epth
bi
oinf
orm
atic
ana
lysi
s of s
eque
nce
data
ava
ilabl
e in
the
liter
atur
e an
d a
new
ly g
ener
ated
V(N
AR)
dat
abas
e. Th
is st
udy
allo
wed
us
to id
entif
y, fo
r the
firs
t tim
e, b
oth
V(N
AR)
-spe
cific
and
V(N
AR)
/Ig
V(L
)/TC
R V
(alp
ha) o
verl
appi
ng h
allm
ark
resi
dues
, whi
ch
are
criti
cal f
or th
e st
ruct
ural
and
func
tiona
l int
egri
ty o
f the
sing
le d
omai
n. In
trig
uing
ly, e
ach
of o
ur d
esig
nate
d V
(NA
R)-s
peci
fic
hallm
arks
alig
n pr
ecis
ely
with
pre
viou
sly
defin
ed m
utat
iona
l ‘co
ld sp
ots’
in n
atur
al n
urse
shar
k cD
NA
sequ
ence
s. Th
ese
findi
ngs
will
aid
futu
re V
(NA
R) e
ngin
eeri
ng a
nd o
ptim
isat
ion
stud
ies t
owar
ds th
e de
velo
pmen
t of V
(NA
R) si
ngle
-dom
ain
prot
eins
as v
iabl
e bi
othe
rape
utic
s.
Fenn
ell
et a
l. 20
10
Yeas
t sur
face
dis
play
Pich
ia p
asto
ris
Gre
en fl
uore
scen
t pr
otei
n
Yeas
t sur
face
dis
play
is a
n effi
cien
t too
l for
isol
atin
g an
d en
gine
erin
g an
tibod
y fr
agm
ents
, bot
h sc
Fv a
nd F
ab. W
e de
scri
be th
e us
e of
pro
tein
dis
play
on
Pich
ia p
asto
ris f
or th
e ra
pid
sele
ctio
n of
cam
elid
ant
ibod
ies c
ompo
sed
only
of h
eavy
cha
ins (
nano
bodi
es)
from
a li
brar
y de
rive
d fr
om a
Ilam
a im
mun
ized
with
Gre
en F
luor
esce
nt P
rote
in. Th
e lib
rary
of n
anob
ody-
codi
ng se
quen
ces w
as
fuse
d to
the
C-t
erm
inal
par
t of t
he S
acch
arom
yces
cer
evis
iae
alph
a-ag
glut
inin
gen
e (S
AG
1) a
nd e
xpre
ssed
in g
lyco
engi
neer
ed
P. p
asto
ris.
A h
igh
effici
ency
tran
sfor
mat
ion
prot
ocol
yie
lded
a li
brar
y of
5 ×
10(
7) c
lone
s. A
bout
80%
of t
he c
lone
s str
ongl
y ex
pres
sed
the
nano
body
fusi
on. N
anob
ody-
disp
layi
ng c
lone
s wer
e ra
pidl
y en
rich
ed b
y flu
ores
cenc
e ac
tivat
ed c
ell s
ortin
g (F
AC
S),
and
GFP
-spe
cific
nan
obod
y-di
spla
ying
clo
nes w
ere
isol
ated
and
equ
ilibr
ium
dis
soci
atio
n co
nsta
nts (
K(d
)) d
eter
min
ed. Th
is
tech
nolo
gy fo
r dis
play
ing
prot
ein
libra
ries
on
P. p
asto
ris e
nabl
es th
e is
olat
ion
and
engi
neer
ing
of a
ntib
ody-
deri
ved
mol
ecul
es in
a
robu
st e
ukar
yotic
exp
ress
ion
host
.
Ryck
aert
et
al.
2010
Surf
ace
disp
lay
Expr
essi
on c
asse
ttes
Lact
obac
illus
par
acas
eiR
otav
irus
A se
ries o
f exp
ress
ion
cass
ette
s whi
ch m
edia
te se
cret
ion
or su
rfac
e di
spla
y of
ant
ibod
y fr
agm
ents
was
stab
ly in
tegr
ated
in th
e ch
rom
o-so
me
of L
acto
baci
llus p
arac
asei
. L. p
arac
asei
pro
duci
ng su
rfac
e-an
chor
ed v
aria
ble
dom
ain
of ll
ama
heav
y ch
ain
(VH
H) (
ARP
1) d
irect
ed
agai
nst r
otav
irus s
how
ed e
ffici
ent b
indi
ng to
rota
viru
s and
pro
tect
ion
in th
e m
ouse
mod
el o
f rot
aviru
s inf
ectio
n.
Mar
tin
et a
l. 20
11
Fung
al e
xpre
ssio
n sy
stem
Asp
ergi
llus
awam
ori
We
repo
rt th
e ex
pres
sion
and
pro
duct
ion
of ll
ama
vari
able
hea
vy-c
hain
ant
ibod
y fr
agm
ents
(V(H
H)s
) by
Asp
ergi
llus a
wam
ori.
Frag
men
ts e
ncod
ing
V(H
H)s
wer
e cl
oned
in a
suita
ble
Asp
ergi
llus e
xpre
ssio
n ve
ctor
and
tran
sfor
man
ts se
cret
ing
V-H
H fr
agm
ents
w
ere
anal
ysed
for i
nteg
rate
d ge
ne c
opy-
num
bers
, mRN
A le
vels
and
pro
tein
pro
duct
ion.
Fun
ctio
nal V
(HH
)s w
ere
dete
cted
in th
e cu
lture
med
ium
, ind
icat
ing
the
feas
ibili
ty o
f pro
duci
ng th
is ty
pe o
f pro
tein
in a
fung
al e
xpre
ssio
n sy
stem
. Sec
rete
d V
(HH
)s w
ere
subj
ecte
d to
(ext
race
llula
r) d
egra
datio
n, w
hich
cou
ld b
e pa
rtia
lly p
reve
nted
by
the
addi
tion
of B
SA to
the
cultu
re m
ediu
m.
Joos
ten
et a
l. 20
05
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
464
Chi
nese
ham
ster
ova
ry
cells
Expr
essi
on v
ecto
rsR
epor
ter
gene
GFP
-fus
ion
prot
ein
Ther
e is
an in
crea
sing
inte
rest
in th
e ap
plic
atio
n of
nan
obod
ies s
uch
as V
HH
in th
e fie
ld o
f the
rapy
and
imag
ing.
In th
e pr
esen
t stu
dy a
st
able
gen
etic
ally
eng
inee
red
cell
line
of C
hine
se h
amst
er o
vary
(CH
O) o
rigin
tran
sfec
ted
usin
g tw
o se
ts o
f exp
ress
ion
vect
ors w
as c
on-
stru
cted
in o
rder
to p
erm
it th
e cy
topl
asm
ic a
nd e
xtra
cellu
lar e
xpre
ssio
n of
sing
le d
omai
n an
tibod
y al
ong
with
gre
en fl
uore
scen
t pro
tein
(G
FP) a
s rep
orte
r gen
e. Th
e qu
ality
of t
he c
onst
ruct
s wer
e ex
amin
ed b
oth
by th
e re
stric
tion
map
as w
ell a
s seq
uenc
e an
alys
is. Th
e ge
ne
tran
sfec
tion
and
prot
ein
expr
essio
n w
as fu
rthe
r exa
min
ed b
y re
vers
e tr
ansc
riptio
n-po
lym
eras
e ch
ain
reac
tion
(RT-
PCR)
. The
tran
sfec
ted
cells
wer
e gr
own
in 2
00 µ
g/m
l hyg
rom
ycin
con
tain
ing
med
ia a
nd th
e st
able
cel
l lin
e ob
tain
ed sh
owed
fluo
resc
ent a
ctiv
ity fo
r mor
e th
an
a pe
riod
of 1
80 d
ays.
The
prod
uctio
n of
fusio
n pr
otei
n w
as a
lso d
etec
ted
by fl
uore
scen
t mic
rosc
opy,
fluor
esce
nt sp
ectr
osco
py a
s wel
l as
by e
nzym
e-lin
ked
imm
unos
orbe
nt a
ssay
(ELI
SA) a
naly
sis. Th
is st
rate
gy a
llow
s a ra
pid
prod
uctio
n of
reco
mbi
nant
fluo
bodi
es in
volv
ing
VH
H, w
hich
can
be
used
in v
ario
us e
xper
imen
ts su
ch a
s im
agin
g an
d de
tect
ion
in w
hich
a p
rimar
y la
bele
d an
tibod
y is
requ
ired.
Bazl
et
al. 2
007
Expr
essi
on in
mou
se
germ
line
Dro
med
ary
tran
sgen
eG
amm
a 2a
H c
hain
In m
atur
e B
cells
of m
ice
and
mos
t mam
mal
s, c
ellu
lar r
elea
se o
f sin
gle
H c
hain
Abs
with
out L
cha
ins i
s pre
vent
ed b
y H
cha
in a
sso-
ciat
ion
with
Ig-s
peci
fic c
hape
rons
in th
e en
dopl
asm
ic re
ticiu
lum
. In
prec
urso
r B c
ells
, how
ever
, sur
face
exp
ress
ion
of µ
-H c
hain
in
the
abse
nce
of su
rrog
ate
and
conv
entio
nal L
cha
in h
as b
een
iden
tified
. Des
pite
this
, Ag-
spec
ific
sing
le H
cha
in Ig
repe
rtoi
res,
usi
ng
µ-, g
amm
a-, e
psilo
n-, o
r alp
ha-H
cha
ins f
ound
in c
onve
ntio
nal A
bs, a
re n
ot p
rodu
ced.
Mor
eove
r, re
mov
al o
f H c
hain
or,
sepa
rate
ly,
L ch
ain
(kap
pa/la
mbd
a) lo
cus c
ore
sequ
ence
s by
gene
targ
etin
g ha
s pre
vent
ed B
cel
l dev
elop
men
t. In
con
tras
t, H
cha
in-o
nly
Abs
ar
e pr
oduc
ed a
bund
antly
in C
amel
idae
as H
2 Ig
G w
ithou
t the
C(H
)l do
mai
n. T
o te
st w
heth
er H
cha
in A
bs c
an b
e pr
oduc
ed in
m
ice,
and
to in
vest
igat
e ho
w th
eir e
xpre
ssio
n aff
ects
B c
ell d
evel
opm
ent,
we
intr
oduc
ed a
rear
rang
ed d
rom
edar
y ga
mm
a 2a
H
chai
n in
to th
e m
ouse
ger
mlin
e. Th
e dr
omed
ary
tran
sgen
e w
as e
xpre
ssed
as a
nat
ural
ly o
ccur
ring
Ag-
spec
ific
disu
lphi
de-li
nked
ho
mod
imer
, whi
ch sh
owed
that
B c
ell d
evel
opm
ent c
an b
e in
stig
ated
by
expr
essi
on o
f sin
gle
H c
hain
s with
out L
cha
ins.
Lym
pho-
cyte
dev
elop
men
t and
B c
ell p
rolif
erat
ion
was
acc
ompl
ishe
d de
spite
the
abse
nce
of L
cha
in fr
om th
e BC
R co
mpl
ex. E
ndog
enou
s Ig
coul
d no
t be
dete
cted
, alth
ough
V(D
)J re
com
bina
tion
and
IgH
/L tr
ansc
ript
ion
was
una
ltere
d. F
urth
erm
ore,
cro
ssin
g th
e dr
om-
edar
y H
cha
in m
ice
with
mic
e de
void
of a
ll C
gen
es d
emon
stra
ted
with
out a
dou
bt th
at a
H c
hain
-onl
y A
b ca
n fa
cilit
ate
B ce
ll de
velo
pmen
t ind
epen
dent
of e
ndog
enou
s Ig
expr
essi
on, s
uch
as µ
- or d
elta
-H c
hain
, at e
arly
dev
elop
men
tal s
tage
s.
Zou
et
al. 2
005
Uni
vers
al h
uman
ized
na
nobo
dy s
caffo
ldN
anob
odie
s, si
ngle
-dom
ain
antig
en-b
indi
ng fr
agm
ents
of c
amel
id-s
peci
fic h
eavy
-cha
in o
nly
antib
odie
s offe
r spe
cial
adv
anta
ges
in th
erap
y ov
er c
lass
ic a
ntib
ody
frag
men
ts b
ecau
se o
f the
ir sm
alle
r siz
e, ro
bust
ness
, and
pre
fere
nce
to ta
rget
uni
que
epito
pes.
A
Nan
obod
y di
ffers
from
a h
uman
hea
vy c
hain
var
iabl
e do
mai
n in
abo
ut te
n am
ino
acid
s spr
ead
all o
ver i
ts su
rfac
e, fo
ur h
allm
ark
Nan
obod
y-sp
ecifi
c am
ino
acid
s in
the
fram
ewor
k-2
regi
on (p
ositi
ons 4
2, 4
9, 5
0, a
nd 5
2), a
nd a
long
er th
ird
antig
en-b
indi
ng lo
op
(H3)
fold
ing
over
this
are
a. F
or th
erap
eutic
app
licat
ions
the
cam
elid
-spe
cific
am
ino
acid
sequ
ence
s in
the
fram
ewor
k ha
ve to
be
mut
ated
to th
eir h
uman
hea
vy c
hain
var
iabl
e do
mai
n eq
uiva
lent
, i.e
. hum
aniz
ed. W
e pe
rfor
med
this
hum
aniz
atio
n ex
erci
se w
ith
Nan
obod
ies o
f the
subf
amily
that
repr
esen
ts c
lose
to 8
0% o
f all
drom
edar
y-de
rive
d N
anob
odie
s and
inve
stig
ated
the
effec
ts o
n an
tigen
affi
nity
, sol
ubili
ty, e
xpre
ssio
n yi
eld,
and
stab
ility
. It i
s dem
onst
rate
d th
at th
e hu
man
izat
ion
of N
anob
ody-
spec
ific
resi
dues
ou
tsid
e fr
amew
ork-
2 ar
e ne
utra
l to
the
Nan
obod
y pr
oper
ties.
Sur
pris
ingl
y, th
e G
lu-4
9 ->
Gly
and
Arg
-50
-> L
eu h
uman
izat
ion
of
hallm
ark
amin
o ac
ids g
ener
ates
a si
ngle
dom
ain
that
is m
ore
stab
le th
ough
pro
babl
y le
ss so
lubl
e. Th
e ot
her f
ram
ewor
k-2
subs
titu-
tions
, Phe
-42
-> V
al a
nd G
ly/A
la-5
2 ->
Trp
, are
det
rim
enta
l for
ant
igen
affi
nity
, due
to a
repo
sitio
ning
of t
he H
3 lo
op a
s sho
wn
by
thei
r cry
stal
stru
ctur
es. Th
ese
insi
ghts
wer
e us
ed to
iden
tify
a so
lubl
e, st
able
, wel
l exp
ress
ed u
nive
rsal
hum
aniz
ed N
anob
ody
scaf
-fo
ld th
at a
llow
s gra
fts o
f ant
igen
-bin
ding
loop
s fro
m o
ther
Nan
obod
ies w
ith tr
ansf
er o
f the
ant
igen
spec
ifici
ty a
nd a
ffini
ty.
Vin
cke
et a
l. 20
09
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
465
Hum
aniz
ed n
anob
odie
sLo
op-g
raft
ing
In v
ivo
imag
ing
Scaff
old
Car
cino
embr
yoni
c an
tige
n
Nan
obod
ies
are
a no
vel t
ype
of im
mun
oglo
bulin
like,
ant
igen
-bin
ding
pro
tein
with
ben
efic
ial p
harm
acol
ogic
and
pha
rmac
oki-
netic
pro
pert
ies
that
are
idea
lly s
uite
d to
targ
etin
g ce
llula
r an
tigen
s fo
r m
olec
ular
imag
ing
or th
erap
eutic
pur
pose
s. H
owev
er,
beca
use
of th
eir
cam
elid
, non
hum
an o
rigi
n, th
e po
ssib
le im
mun
ogen
icity
of N
anob
odie
s w
hen
used
in th
e cl
inic
is a
con
cern
. H
ere
we
pres
ent a
new
str
ateg
y to
qui
ckly
gen
erat
e hu
man
ized
Nan
obod
ies
for
mol
ecul
ar im
agin
g pu
rpos
es. M
etho
ds: W
e ge
netic
ally
gra
fted
the
antig
en-b
indi
ng lo
ops
of N
bCEA
5, a
Nan
obod
y w
ith s
peci
ficity
for
the
colo
n ca
rcin
oma
mar
ker
carc
i-no
embr
yoni
c an
tigen
(CEA
), on
to th
e fr
amew
ork
of a
hum
aniz
ed N
anob
ody
scaf
fold
. Thi
s sc
affo
ld h
as b
een
prev
ious
ly c
hara
c-te
rize
d in
our
labo
rato
ry a
s a
stab
le N
anob
ody
that
can
ser
ve a
s a
univ
ersa
l loo
p ac
cept
or fo
r an
tigen
-bin
ding
loop
s fr
om d
onor
N
anob
odie
s an
d ha
s be
en a
dditi
onal
ly m
utat
ed a
t abo
ut 1
0 cr
ucia
l sur
face
-exp
osed
site
s to
rese
mbl
e th
e se
quen
ce o
f hum
an
vari
able
imm
unog
lobu
lin d
omai
ns. T
he 3
reco
mbi
nant
Nan
obod
ies
(NbC
EA5,
hum
aniz
ed s
caffo
ld, a
nd h
uman
ized
CEA
5 gr
aft)
w
ere
prod
uced
in b
acte
ria
and
puri
fied.
Unl
abel
ed a
nd (9
9m)T
c-la
bele
d N
anob
odie
s w
ere
bioc
hem
ical
ly c
hara
cter
ized
in v
itro
and
test
ed a
s pr
obes
for
SPEC
T/C
T o
f xen
ogra
fted
tum
ors.
Res
ults
: The
suc
cess
of l
oop-
graf
ting
was
con
firm
ed b
y co
mpa
ring
th
ese
Nan
obod
ies
for
thei
r ca
paci
ty to
reco
gniz
e so
lubl
e C
EA p
rote
in in
enz
yme-
linke
d im
mun
osor
bent
ass
ay a
nd b
y su
rfac
e pl
asm
on re
sona
nce
and
to b
ind
to C
EA-p
ositi
ve L
S174
T c
olon
car
cino
ma
cells
and
CEA
-tra
nsfe
cted
but
not
unt
rans
fect
ed C
hi-
nese
ham
ster
ova
ry c
ells
in fl
ow c
ytom
etry
. Spe
cific
ity o
f bin
ding
was
con
firm
ed b
y co
mpe
titio
n st
udie
s. A
ll N
anob
odie
s w
ere
heat
-sta
ble,
cou
ld b
e ef
ficie
ntly
labe
led
with
(99m
)Tc,
and
reco
gniz
ed b
oth
solu
ble
and
mem
bran
e-bo
und
CEA
pro
tein
in b
ind-
ing
stud
ies.
Fin
ally
, bio
dist
ribu
tion
expe
rim
ents
wer
e pe
rfor
med
with
intr
aven
ousl
y in
ject
ed (9
9m)T
c-la
bele
d N
anob
odie
s in
LS
174T
tum
or-b
eari
ng m
ice
usin
g pi
nhol
e SP
ECT
/mic
ro-C
T. T
hese
in v
ivo
expe
rim
ents
reve
aled
spe
cific
ity o
f tum
or ta
rget
ing
and
rapi
d re
nal c
lear
ance
for
all N
anob
odie
s, w
ith lo
w s
igna
ls in
all
orga
ns b
esid
es th
e ki
dney
s. C
oncl
usio
n: T
his
stud
y sh
ows
the
pote
ncy
of a
ntig
en-b
indi
ng lo
op-g
raft
ing
to e
ffic
ient
ly g
ener
ate
hum
aniz
ed N
anob
odie
s th
at re
tain
thei
r ta
rget
ing
capa
ci-
ties
for
noni
nvas
ive
in v
ivo
imag
ing
of tu
mor
s.
Vane
y-ck
en e
t al
. 201
0
Gra
ftin
gH
apte
nM
etho
trex
ate
Cry
stal
str
uctu
re
anal
ysis
Whi
le s
ever
al a
nti-
hapt
en V
HH
s ha
ve b
een
gene
rate
d, li
ttle
is k
now
n re
gard
ing
the
unde
rlyi
ng s
truc
tura
l and
ther
mod
y-na
mic
bas
is fo
r ha
pten
reco
gniti
on. H
ere,
an
anti-
met
hotr
exat
e V
HH
(ant
i-M
TX
VH
H) w
as g
ener
ated
usi
ng g
raft
ing
met
hods
w
here
by th
e th
ree
com
plem
enta
rity
det
erm
inin
g re
gion
s (C
DR
s) w
ere
inse
rted
ont
o an
exi
stin
g V
HH
fram
ewor
k. T
herm
ody-
nam
ic a
naly
sis
of th
e an
ti-M
TX
VH
H C
DR1
–3 G
raft
reve
aled
a m
icro
mol
ar b
indi
ng a
ffin
ity, w
hile
the
crys
tal s
truc
ture
of t
he
com
plex
reve
aled
a s
omew
hat s
urpr
isin
g no
ncan
onic
al b
indi
ng s
ite w
hich
invo
lved
MT
X tu
nnel
ing
unde
r th
e C
DR1
loop
. Due
to
the
clos
e pr
oxim
ity o
f MT
X to
CD
R4, a
non
hype
rvar
iabl
e lo
op, t
he C
DR4
loop
seq
uenc
e w
as s
ubse
quen
tly in
trod
uced
into
th
e C
DR1
–3 g
raft
, whi
ch re
sulte
d in
a d
ram
atic
100
0-fo
ld in
crea
se in
the
bind
ing
affin
ity. C
ryst
al s
truc
ture
ana
lysi
s of
bot
h th
e fr
ee a
nd c
ompl
ex a
nti-
MT
X C
DR1
–4 g
raft
reve
aled
CD
R4 p
lays
a s
igni
fican
t rol
e in
bot
h in
term
olec
ular
con
tact
s an
d bi
ndin
g si
te c
onfo
rmat
ion
that
app
ear
to c
ontr
ibut
e to
war
d hi
gh a
ffin
ity b
indi
ng. A
dditi
onal
ly, t
he a
nti-
MT
X V
HH
pos
sess
ed re
lativ
ely
high
spe
cific
ity fo
r M
TX
ove
r cl
osel
y re
late
d co
mpo
unds
am
inop
teri
n an
d fo
late
, dem
onst
ratin
g th
at V
HH
dom
ains
are
cap
able
of
bin
ding
low
-mol
ecul
ar w
eigh
t lig
ands
with
hig
h af
finity
and
spe
cific
ity, d
espi
te th
eir
redu
ced
inte
rfac
e.
Fann
ing
and
Hor
n 20
11
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
466
Gra
ftin
gTo
bacc
oLy
sozy
me
‘Uni
vers
al’ n
anob
ody
fram
ewor
k
Her
e w
e de
scri
be h
igh-
leve
l exp
ress
ion,
in N
icot
iana
ben
tham
iana
, of t
hree
ver
sion
s of a
n an
ti-he
n eg
g w
hite
lyso
zym
e (H
EWL)
na
nobo
dy w
hich
incl
ude
the
orig
inal
VH
H fr
om a
n im
mun
ized
libr
ary
(cA
bLys
3), a
cod
on-o
ptim
ized
der
ivat
ive,
and
a c
odon
-op
timiz
ed h
ybri
d na
nobo
dy c
ompr
isin
g th
e C
DRs
of c
AbL
ys3
graf
ted
onto
an
alte
rnat
ive
‘uni
vers
al’ n
anob
ody
fram
ewor
k. H
is6-
an
d St
repI
I-ta
gged
der
ivat
ives
of e
ach
nano
body
wer
e ta
rget
ed fo
r acc
umul
atio
n in
the
cyto
plas
m, c
hlor
opla
st a
nd a
popl
ast u
sing
di
ffere
nt p
re-s
eque
nces
. Whe
n ta
rget
ed to
the
apop
last
, int
act f
unct
iona
l nan
obod
ies a
ccum
ulat
ed a
t an
exce
ptio
nally
hig
h le
vel
(up
to 3
0% to
tal l
eaf p
rote
in),
dem
onst
ratin
g th
e gr
eat p
oten
tial o
f pla
nts a
s a n
anob
ody
prod
uctio
n sy
stem
.
Teh
and
Kav
a-
nagh
20
10
Epid
erm
al g
row
th
fact
or r
ecep
tor
Pent
abod
yLo
ng s
erum
hal
f lif
eFc
frag
men
t
In th
e se
arch
for b
ette
r ant
ibod
y fo
rmat
s for
in v
ivo
imag
ing
and/
or th
erap
y of
can
cer,
thre
e ty
pes o
f sdA
b-ba
sed
mol
ecul
es
dire
cted
aga
inst
epi
derm
al g
row
th fa
ctor
rece
ptor
(EG
ER) w
ere
cons
truc
ted,
cha
ract
eriz
ed a
nd te
sted
. Ele
ven
sdA
bs w
ere
isol
ated
fr
om a
pha
ge d
ispl
ay li
brar
y co
nstr
ucte
d fr
om th
e sd
Ab
repe
rtoi
re o
f a ll
ama
imm
uniz
ed w
ith a
var
iant
of E
GFR
. A p
enta
mer
ic
sdA
b, o
r pen
tabo
dy, V
2C-E
G2
was
con
stru
cted
by
fusi
ng o
ne o
f the
sdA
bs, E
G2,
to a
pen
tam
eriz
atio
n pr
otei
n do
mai
n. A
chi
mer
ic
HC
Ab
(cH
CA
b), E
G2-
hFc,
was
con
stru
cted
by
fusi
ng E
G2
to th
e fr
agm
ent c
ryst
alliz
able
(Fc)
of h
uman
IgG
1. W
here
as E
G2
and
V2C
-EG
2 lo
caliz
ed m
ainl
y in
the
kidn
eys a
fter
i.v.
inje
ctio
n, E
G2-
hFc
exhi
bite
d ex
celle
nt tu
mor
acc
umul
atio
n, a
nd th
is w
as la
rgel
y at
trib
uted
to it
s lon
g se
rum
hal
f life
, whi
ch is
com
para
ble
to th
at o
f IgG
s. Th
e m
oder
ate
size
(sim
ilar t
o 80
kD
a) a
nd in
tact
hum
an
Fc m
ake
HC
Abs
a u
niqu
e an
tibod
y fo
rmat
whi
ch m
ay o
utpe
rfor
m w
hole
IgG
s as i
mag
ing
and
ther
apeu
tic re
agen
ts.
Bell
et
al. 2
010
Uni
vers
al fr
amew
ork
Gra
ftin
gC
him
era
Cam
el si
ngle
-dom
ain
antib
ody
frag
men
ts (V
HH
s) a
re p
rom
isin
g to
ols i
n nu
mer
ous b
iote
chno
logi
cal a
nd m
edic
al a
pplic
atio
ns.
How
ever
, som
e co
nditi
ons u
nder
whi
ch a
ntib
odie
s are
use
d ar
e so
dem
andi
ng th
at th
ey c
an b
e m
et b
y on
ly th
e m
ost r
obus
t VH
Hs.
A u
nive
rsal
fram
ewor
k off
erin
g th
e re
quire
d pr
oper
ties f
or u
se in
var
ious
app
licat
ions
(e.g
. as i
ntra
body
, as p
robe
in b
iose
nsor
s or o
n m
icro
-arr
ays)
is h
ighl
y va
luab
le a
nd m
ight
be
furt
her i
mpl
emen
ted
whe
n em
ploy
men
t of V
HH
s in
hum
an th
erap
y is
env
isag
ed. W
e id
entifi
ed th
e V
HH
fram
ewor
k of
cA
bBC
II10
as a
pot
entia
l can
dida
te, u
sefu
l for
the
exch
ange
of a
ntig
en sp
ecifi
citie
s by
com
plem
en-
tari
ty d
eter
min
ing
regi
on (C
DR)
gra
fting
. Due
to th
e la
rge
num
ber o
f CD
RH lo
op st
ruct
ures
pre
sent
on
VH
Hs,
this
gra
fting
tech
-ni
que
was
exp
ecte
d to
be
rath
er u
npre
dict
able
. Non
ethe
less
, the
pla
stic
ity o
f the
cA
bBC
II10
fram
ewor
k al
low
s suc
cess
ful t
rans
fer o
f an
tigen
spec
ifici
ty fr
om d
onor
VH
Hs o
nto
its sc
affol
d. Th
e cA
bBC
II10
was
cho
sen
esse
ntia
lly fo
r its
hig
h le
vel o
f sta
bilit
y (4
7 kJ
/mol
, go
od e
xpre
ssio
n le
vel (
5 m
g/l i
n E.
col
i) an
d its
abi
lity
to b
e fu
nctio
nal i
n th
e ab
senc
e of
the
cons
erve
d di
sulfi
de b
ond.
All
five
chim
e-ra
s gen
erat
ed b
y gr
aftin
g C
DR-
Hs,
from
don
or V
HH
s bel
ongi
ng to
subf
amily
2 th
at e
ncom
pass
75%
of a
ll an
tigen
-spe
cific
VH
Hs,
on
the
fram
ewor
k of
cA
bBC
II10
wer
e fu
nctio
nal a
nd g
ener
ally
had
an
incr
ease
d th
erm
odyn
amic
stab
ility
. The
graf
ting
of C
DR-
H lo
ops
from
VH
Hs b
elon
ging
to o
ther
subf
amili
es re
sulte
d in
chi
mer
as o
f red
uced
ant
igen
-bin
ding
cap
acity
.
Saer
ens
et a
l. 20
05
VH
H-S
NA
P co
nstr
uct
Erv1
p su
lfhy
dryl
ox
idas
e
Cam
elid
ae si
ngle
dom
ain
antib
odie
s (V
HH
s) h
ave
stru
ctur
al a
nd b
indi
ng fe
atur
es th
at re
nder
them
suita
ble
alte
rnat
ives
to c
on-
vent
iona
l IgG
ant
ibod
ies.
VH
Hs a
re u
sual
ly e
asie
r to
prod
uce
as re
com
bina
nt p
rote
ins t
han
othe
r ant
ibod
y fr
agm
ents
. How
ever
, fo
r som
e of
the
biot
echn
olog
ical
app
licat
ions
for w
hich
they
hav
e be
en p
ropo
sed,
such
as i
mm
unoc
hrom
atog
raph
y an
d as
sist
ed-
crys
tallo
grap
hy, l
arge
am
ount
s of p
urifi
ed a
ntib
odie
s are
nec
essa
ry, w
here
as so
me
VH
H-f
usio
ns w
ith c
omm
on ta
gs su
ch a
s GFP
an
d SN
AP
are
poor
ly e
xpre
ssed
in th
e ba
cter
ial p
erip
lasm
. Her
e w
e ha
ve sh
own
that
the
co-e
xpre
ssio
n of
Erv
1p su
lfhyd
ryl o
xida
se
resu
lted
in a
n as
toni
shin
g yi
eld
incr
ease
of V
HH
-SN
AP
cons
truc
ts e
xpre
ssed
in th
e ba
cter
ial c
ytop
lasm
. The
resu
lting
reco
m-
bina
nt a
ntib
odie
s wer
e al
so m
ore
stab
le th
an th
e an
tibod
ies p
rodu
ced
usin
g th
e sa
me
plas
mid
, but
in w
ild-t
ype
bact
eria
. Usi
ng
this
app
roac
h, it
was
pos
sibl
e to
obt
ain
tens
of m
illig
ram
s of p
urifi
ed fu
sion
ant
ibod
ies u
sing
a b
asic
flas
k fe
rmen
tatio
n pr
otoc
ol.
Ther
efor
e, th
e de
scri
bed
met
hod
repr
esen
ts a
val
id so
lutio
n to
pro
duce
inex
pens
ivel
y la
rge
amou
nts o
f sin
gle
dom
ain
antib
odie
s fo
r in
vitr
o ap
plic
atio
ns a
nd w
e ex
pect
it w
ill b
e su
itabl
e fo
r the
pro
duct
ion
of o
ther
ant
ibod
y fr
agm
ents
.
Vegg
iani
an
d de
M
arco
20
11
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
467
Tabl
e 5.
Cha
ract
eris
tic
prop
erti
es o
f sin
gle-
dom
ain
anti
body
frag
men
ts
Blo
od-b
rain
bar
rier
pe
rmea
bilit
yTr
ypan
osom
a br
ucei
Surf
ace
glyc
opro
tein
V
SG
Expe
rim
enta
l app
roac
h: W
e ha
ve a
sses
sed
the
bloo
d-br
ain
barr
ier p
erm
eabi
lity
of N
b_A
n33,
a n
anob
ody
agai
nst t
he T
rypa
noso
ma
bruc
ei b
ruce
i var
iant
-spe
cific
surf
ace
glyc
opro
tein
(VSG
). Th
is a
naly
sis w
as p
erfo
rmed
in h
ealth
y ra
ts a
nd in
rats
that
wer
e in
the
ence
phal
itic
stag
e of
Afr
ican
tryp
anos
omia
sis u
sing
intr
acer
ebra
l mic
rodi
alys
is, s
ingl
e ph
oton
em
issi
on c
ompu
ted
tom
ogra
phy
(SPE
CT
) or a
com
bina
tion
of b
oth
met
hodo
logi
es. Th
is e
nabl
ed th
e qu
antifi
catio
n of
unl
abel
led
and
Tc-9
9m-la
belle
d na
nobo
dies
us
ing,
resp
ectiv
ely,
a se
nsiti
ve V
SG-b
ased
nan
obod
y-de
tect
ion
ELIS
A, r
adio
activ
ity m
easu
rem
ent i
n co
llect
ed m
icro
dial
ysat
es a
nd
SPEC
T im
age
anal
ysis
. Key
resu
lts: Th
e co
mbi
ned
read
-out
met
hodo
logi
es sh
owed
that
Nb_
An3
3 w
as d
etec
ted
in th
e br
ain
of h
ealth
y ra
ts fo
llow
ing
i.v. i
njec
tion,
infla
mm
atio
n-in
duce
d da
mag
e to
the
bloo
d-br
ain
barr
ier,
as in
the
late
enc
epha
litic
stag
e of
tryp
anos
o-m
iasi
s, si
gnifi
cant
ly in
crea
sed
the
effici
ency
of p
assa
ge o
f the
nan
obod
y th
roug
h th
is b
arri
er. C
ompl
emen
ting
SPEC
T a
naly
ses w
ith
intr
acer
ebra
l mic
rodi
alys
is im
prov
ed a
naly
sis o
f bra
in d
ispo
sitio
n. Th
ere
is c
lear
val
ue in
ass
essi
ng p
enet
ratio
n of
the
bloo
d-br
ain
bar-
rier
by
mon
oval
ent n
anob
odie
s in
mod
els o
f CN
S in
flam
mat
ion.
Our
dat
a al
so su
gges
t tha
t rap
id c
lear
ance
from
blo
od m
ight
ham
per
effici
ent t
arge
ting
of sp
ecifi
c na
nobo
dies
to th
e C
NS.
Con
clus
ions
and
impl
icat
ions
: Nan
obod
ies c
an e
nter
the
brai
n pa
renc
hym
a fr
om
the
syst
emic
circ
ulat
ion,
esp
ecia
lly in
pat
holo
gica
l con
ditio
ns w
here
the
bloo
d-br
ain
barr
ier i
nteg
rity
is c
ompr
omis
ed.
Cal
jon
et
al. 2
012
Bet
a-am
yloi
dTr
ansm
igra
tion
effi
cien
cyA
lzhe
imer
’s di
seas
eC
ereb
ral a
myl
oid
angi
-op
athy
Prev
ious
ly se
lect
ed a
myl
oid
beta
reco
gniz
ing
heav
y ch
ain
antib
ody
frag
men
ts (V
HH
) affi
nity
bin
ders
der
ived
from
the
Cam
elid
he
avy
chai
n an
tibod
y re
pert
oire
wer
e te
sted
for t
heir
pro
pens
ity to
cro
ss th
e bl
ood-
brai
n ba
rrie
r (BB
B) u
sing
an
esta
blis
hed
in v
itro
BBB
co-c
ultu
re sy
stem
. Of a
ll te
sted
VH
H, n
i3A
show
ed h
ighe
st tr
ansm
igra
tion
effici
ency
whi
ch is
, in
part
, fac
ilita
ted
by a
thre
e am
ino
acid
subs
titut
ions
in it
s N-t
erm
inal
dom
ain.
Add
ition
al st
udie
s ind
icat
ed th
at th
e m
echa
nism
of t
rans
cellu
lar p
assa
ge o
f ni
3A is
by
activ
e tr
ansp
ort.
As V
HH
ni3
A c
ombi
nes t
he a
bilit
y to
reco
gniz
e am
yloi
d be
ta a
nd to
cro
ss th
e BB
B, it
has
pot
entia
l as
a to
ol fo
r non
-inva
sive
in v
ivo
imag
ing
and
as e
ffici
ent l
ocal
dru
g ta
rget
ing
moi
ety
in p
atie
nts s
uffer
ing
from
cer
ebra
l am
yloi
dosi
s su
ch a
s Alz
heim
er’s
dise
ase
(AD
) and
cer
ebra
l am
yloi
d an
giop
athy
(CA
A).
Rutg
ers
et a
l. 20
11
Blo
od-b
rain
bar
rier
Tran
scyt
osis
Endo
cyto
sis
Cer
ebra
l end
othe
lial
cells
Puta
tive
rec
epto
r
Ant
ibod
ies a
gain
st re
cept
ors t
hat u
nder
go tr
ansc
ytos
is a
cros
s the
blo
od-b
rain
bar
rier
(BBB
) hav
e be
en u
sed
as v
ecto
rs to
targ
et
drug
s or t
hera
peut
ic p
eptid
es in
to th
e br
ain.
We
have
rece
ntly
dis
cove
red
a no
vel s
ingl
e do
mai
n an
tibod
y, FC
5, w
hich
tran
smi-
grat
es a
cros
s hum
an c
ereb
ral e
ndot
helia
l cel
ls in
vitr
o an
d th
e BB
B in
viv
o. Th
e pu
rpos
e of
this
stud
y w
as to
cha
ract
eriz
e m
echa
-ni
sms o
f FC
5 en
docy
tosi
s and
tran
scyt
osis
acr
oss t
he B
BB a
nd it
s put
ativ
e re
cept
or o
n hu
man
bra
in e
ndot
helia
l cel
ls. Th
e tr
ans-
port
of F
C5
acro
ss h
uman
bra
in e
ndot
helia
l cel
ls w
as p
olar
ized
, cha
rge
inde
pend
ent a
nd te
mpe
ratu
re d
epen
dent
, sug
gest
ing
a re
cept
or-m
edia
ted
proc
ess.
FC
5 ta
ken
up b
y hu
man
bra
in e
ndot
helia
l cel
ls c
o-lo
caliz
ed w
ith c
lath
rin
but n
ot w
ith c
aveo
lin-1
by
imm
unoc
hem
istr
y an
d w
as d
etec
ted
in c
lath
rin-
enri
ched
subc
ellu
lar f
ract
ions
by
wes
tern
blo
t. Th
e tr
anse
ndot
helia
l mig
ratio
n of
FC
5 w
as re
duce
d by
inhi
bito
rs o
f cla
thri
n-m
edia
ted
endo
cyto
sis,
K+
depl
etio
n an
d ch
lorp
rom
azin
e, b
ut w
as in
sens
itive
to c
aveo
lae
inhi
bito
rs, fi
lipin
, nys
tatin
or m
ethy
l-bet
a-cy
clod
extr
in. F
ollo
win
g in
tern
aliz
atio
n, F
C5
was
targ
eted
to e
arly
end
osom
es, b
ypas
sed
late
end
osom
es/ly
soso
mes
and
rem
aine
d in
tact
aft
er tr
ansc
ytos
is. Th
e tr
ansc
ytos
is p
roce
ss w
as in
hibi
ted
by a
gent
s tha
t affe
ct a
ctin
cy
tosk
elet
on o
r int
race
llula
r sig
nalin
g th
roug
h PI
3-ki
nase
. Pre
trea
tmen
t of h
uman
bra
in e
ndot
helia
l cel
ls w
ith w
heat
germ
agg
lu-
tinin
, sia
lic a
cid,
alp
ha(2
,3)-
neur
amin
idas
e or
Maa
ckia
am
uren
sis a
gglu
tinin
that
reco
gniz
es a
lpha
(2,3
)-, b
ut n
ot w
ith S
ambu
cus
nigr
a ag
glut
inin
that
reco
gniz
es a
lpha
(2,6
)-si
alyl
gala
ctos
yl re
sidu
es, s
igni
fican
tly re
duce
d FC
5 tr
ansc
ytos
is. F
C5
faile
d to
reco
gniz
e br
ain
endo
thel
ial c
ells
-der
ived
lipi
ds, s
ugge
stin
g th
at it
bin
ds lu
min
al a
lpha
(2,3
)-si
alog
lyco
prot
ein
rece
ptor
whi
ch tr
igge
rs c
lath
rin-
med
iate
d en
docy
tosi
s. Th
is p
utat
ive
rece
ptor
may
be
a ne
w ta
rget
for d
evel
opin
g br
ain-
targ
etin
g dr
ug d
eliv
ery
vect
ors.
Abu
lrob
et
al.
2005
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
468
Ther
mal
sta
bilit
yR
esis
tanc
e to
tryp
sin
and
extr
eme
pHC
lost
ridi
um d
iffici
leTo
xin
AO
ral a
dmin
istr
atio
n
The
extr
eme
pH a
nd p
rote
ase-
rich
env
iron
men
t of t
he u
pper
gas
troi
ntes
tinal
trac
t is a
maj
or o
bsta
cle
faci
ng o
rally
-adm
inis
tere
d pr
otei
n th
erap
eutic
s, in
clud
ing
antib
odie
s. Th
roug
h pr
otei
n en
gine
erin
g, se
vera
l Clo
stri
dium
diffi
cile
toxi
n A
-spe
cific
hea
vy c
hain
an
tibod
y va
riab
le d
omai
ns (V
(H)H
s) w
ere
expr
esse
d w
ith a
n ad
ditio
nal d
isul
fide
bond
by
intr
oduc
ing
Ala
/Gly
54C
ys a
nd Il
e78C
ys
mut
atio
ns. M
utan
t ant
ibod
ies w
ere
com
pare
d to
thei
r wild
-typ
e co
unte
rpar
ts w
ith re
spec
t to
expr
essi
on y
ield
, non
-agg
rega
tion
stat
us, a
ffini
ty fo
r tox
in A
, cir
cula
r dic
hroi
sm (C
D) s
truc
tura
l sig
natu
res,
ther
mal
stab
ility
, pro
teas
e re
sist
ance
, and
toxi
n A
-neu
-tr
aliz
ing
capa
city
. The
mut
ant V
(H)H
s wer
e fo
und
to b
e w
ell e
xpre
ssed
, alth
ough
with
low
er y
ield
s com
pare
d to
wild
-typ
e co
un-
terp
arts
, wer
e no
n-ag
greg
atin
g m
onom
ers,
reta
ined
low
nM
affi
nity
for t
oxin
A, a
lbei
t the
maj
ority
show
ed so
mew
hat r
educ
ed
affini
ty c
ompa
red
to w
ild-t
ype
coun
terp
arts
, and
wer
e ca
pabl
e of
in v
itro
toxi
n A
neu
tral
izat
ion
in c
ell-b
ased
ass
ays.
Far
-UV
and
ne
ar-U
V C
D sp
ectr
osco
py c
onsi
sten
tly sh
owed
shift
s in
peak
inte
nsity
and
sele
ctiv
e pe
ak m
inim
a fo
r wild
-typ
e an
d m
utan
t V(H
)H
pair
s; ho
wev
er, t
he o
vera
ll C
D p
rofil
e re
mai
ned
very
sim
ilar.
A si
gnifi
cant
incr
ease
in th
e th
erm
al u
nfol
ding
mid
poin
t tem
pera
ture
w
as o
bser
ved
for a
ll m
utan
ts a
t bot
h ne
utra
l and
aci
dic
pH. D
iges
tion
of th
e V
(H)H
s with
the
maj
or g
astr
oint
estin
al p
rote
ases
, at
biol
ogic
ally
rele
vant
con
cent
ratio
ns, r
evea
led
a si
gnifi
cant
incr
ease
in p
epsi
n re
sist
ance
for a
ll m
utan
ts a
nd a
n in
crea
se in
chy
mot
-ry
psin
resi
stan
ce fo
r the
maj
ority
of m
utan
ts. M
utan
t V(H
)H tr
ypsi
n re
sist
ance
was
sim
ilar t
o th
at o
f wild
-typ
e V
(H)H
s, a
lthou
gh
the
tryp
sin
resi
stan
ce o
f one
V(H
)H m
utan
t was
sign
ifica
ntly
redu
ced.
Ther
efor
e, th
e in
trod
uctio
n of
a se
cond
dis
ulfid
e bo
nd in
th
e hy
drop
hobi
c co
re n
ot o
nly
incr
ease
s V(H
)H th
erm
al st
abili
ty a
t neu
tral
pH
, as p
revi
ousl
y sh
own,
but
als
o re
pres
ents
a g
ener
ic
stra
tegy
to in
crea
se V
(H)H
stab
ility
at l
ow p
H a
nd im
part
pro
teas
e re
sist
ance
, with
onl
y m
inor
per
turb
atio
ns in
targ
et b
indi
ng
affini
ties.
Thes
e ar
e al
l des
irab
le c
hara
cter
istic
s for
the
desi
gn o
f pro
tein
-bas
ed o
ral t
hera
peut
ics.
Hus
sack
et
al.
2011
a
Hea
t den
atur
atio
nEp
ider
mal
gro
wth
fact
or
rece
ptor
Pich
ia p
asto
ris
Mal
igna
ncy
It h
as b
een
show
n th
at, i
n co
ntra
st w
ith c
onve
ntio
nal a
ntib
ody
frag
men
ts, t
he v
aria
ble
dom
ains
of t
hese
hea
vy-c
hain
ant
ibod
ies
are
func
tiona
l at o
r aft
er e
xpos
ure
to h
igh
tem
pera
ture
s. In
the
pres
ent s
tudy
, the
VH
H (v
aria
ble
dom
ain
of h
eavy
-cha
in a
ntib
ody)
ca
mel
ant
ibod
y w
as su
bclo
ned
into
vec
tor P
picz
c an
d ex
pres
sed
in P
ichi
a pa
stor
is. O
RBI-
83 V
HH
ant
ibod
y re
cogn
izes
the
exte
rnal
do
mai
n of
the
mut
ant E
GFR
[EG
F (e
pide
rmal
gro
wth
fact
or) r
ecep
tor]
, EG
FR V
III.
This
tum
our-
spec
ific
antig
en is
liga
nd-i
nde-
pend
ent,
cont
ains
a c
onst
itutiv
ely
activ
e ty
rosi
ne k
inas
e do
mai
n an
d ha
s bee
n sh
own
to b
e pr
esen
t in
a nu
mbe
r of h
uman
mal
ig-
nanc
ies.
We
repo
rt h
ere
that
, alth
ough
exp
ress
ion
from
P. p
asto
ris r
esul
ted
in a
sign
ifica
ntly
incr
ease
d le
vel o
f exp
ress
ion
of th
e an
ti-EG
FR V
III V
HH
ant
ibod
ies c
ompa
red
with
Esc
heri
chia
, thi
s ant
ibod
y se
lect
ivel
y bo
und
to th
e EG
FR V
III p
eptid
e an
d re
acte
d sp
ecifi
cally
with
the
imm
unoa
ffini
ty-p
urifi
ed a
ntig
en fr
om n
on-s
mal
l-cel
l lun
g ca
ncer
. Fur
ther
mor
e, th
erm
al d
enat
urat
ion
stab
il-ity
and
CD
spec
tra
anal
ysis
of t
he C
amel
us b
actr
ianu
s (Ba
ctri
an c
amel
) VH
H a
nd h
eavy
-cha
in a
ntib
odie
s at d
iffer
ent t
empe
ratu
re
prov
ed re
vers
ibili
ty a
nd b
indi
ng a
ctiv
ity a
fter
hea
t den
atur
atio
n. O
ur re
sults
indi
cate
that
the
P. p
asto
ris e
xpre
ssio
n sy
stem
may
be
usef
ul fo
r the
exp
ress
ion
of c
amel
sing
le d
omai
n an
tibod
y an
d th
e ab
ility
of t
he e
xpre
ssed
pro
tein
to re
vers
ibly
mel
t with
out a
ggre
-ga
tion,
allo
win
g it
to re
gain
bin
ding
act
ivity
aft
er h
eat d
enat
urat
ion.
Om
idfa
r et
al.
2007
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
469
Den
atur
ing
agen
tsC
elia
c pa
tien
tsPr
olam
inG
liadi
nEL
ISA
Food
inte
nded
for c
elia
c pa
tient
s’ co
nsum
ptio
n m
ust b
e an
alyz
ed fo
r the
pre
senc
e of
toxi
c pr
olam
ins u
sing
hig
h de
lect
abili
ty te
sts.
Thou
gh 6
0% e
than
ol is
the
mos
t com
mon
ly u
sed
solv
ent f
or p
rola
min
s ext
ract
ion,
2-m
erca
ptoe
than
ol (2
-ME)
and
gua
nidi
nium
chl
o-ri
de (G
uHC
l) ca
n be
add
ed to
incr
ease
pro
tein
reco
very
. How
ever
, eth
anol
and
den
atur
ing
agen
ts in
terf
ere
with
ant
igen
reco
gniti
on
whe
n co
nven
tiona
l ant
ibod
ies a
re u
sed.
In th
e pr
esen
t wor
k, a
new
met
hod
for g
liadi
ns q
uant
ifica
tion
is sh
own.
The
met
hod
is b
ased
on
the
sele
ctio
n of
llam
a si
ngle
dom
ain
antib
ody
frag
men
ts a
ble
to o
pera
te u
nder
den
atur
ing
cond
ition
s. Si
x ou
t of 2
8 V
HH
-pha
ges
obta
ined
reta
ined
thei
r bin
ding
cap
acity
in 1
5% e
than
ol. S
elec
ted
clon
es p
rese
nted
a lo
ng C
DR3
regi
on c
onta
inin
g tw
o ad
ditio
nal
cyst
eine
s tha
t cou
ld b
e re
spon
sibl
e fo
r the
hig
her s
tabi
lity.
One
of t
he c
lone
s (na
med
VH
H26
) was
fully
ope
rativ
e in
the
pres
ence
of
15%
eth
anol
, 0.5
% 2
-ME,
and
0.5
M G
uHC
l. C
aptu
re E
LISA
usi
ng V
HH
26 w
as a
ble
to d
etec
t glia
dins
in sa
mpl
es sh
own
as n
egat
ives
by
con
vent
iona
l ELI
SA. Th
eref
ore,
this
new
stra
tegy
app
ears
as a
n ex
celle
nt p
latfo
rm fo
r qua
ntita
tive
dete
rmin
atio
n of
pro
tein
s or a
ny
othe
r im
mun
ogen
ic c
ompo
und,
in th
e pr
esen
ce o
f den
atur
ing
agen
ts, w
hen
spec
ific
reco
gniti
on u
nits
with
hig
h st
abili
ty a
re re
quire
d.
Don
a et
al
. 201
0
Ente
roto
xin
BD
enat
ured
pro
tein
re
fold
ing
Dia
gnos
tic
assa
y
Back
grou
nd: C
amel
ids a
nd sh
arks
pos
sess
a u
niqu
e su
bcla
ss o
f ant
ibod
ies c
ompr
ised
of o
nly
heav
y ch
ains
. The
antig
en b
ind-
ing
frag
men
ts o
f the
se u
niqu
e an
tibod
ies c
an b
e cl
oned
and
exp
ress
ed a
s sin
gle
dom
ain
antib
odie
s (sd
Abs
). Th
e ab
ility
of t
hese
sm
all a
ntig
en-b
indi
ng m
olec
ules
to re
fold
aft
er h
eatin
g to
ach
ieve
thei
r ori
gina
l str
uctu
re, a
s wel
l as t
heir
dim
inut
ive
size
, mak
es
them
att
ract
ive
cand
idat
es fo
r dia
gnos
tic a
ssay
s. R
esul
ts: H
ere
we
desc
ribe
the
isol
atio
n of
an
sdA
b ag
ains
t Sta
phyl
occo
cus a
ureu
s en
tero
toxi
n B
(SEB
). Th
e cl
one,
A3,
was
foun
d to
hav
e hi
gh a
ffini
ty (K
d =
75 p
M) a
nd g
ood
spec
ifici
ty fo
r SEB
, sho
win
g no
cro
ss
reac
tivity
to re
late
d m
olec
ules
such
as S
taph
yloc
occa
l ent
erot
oxin
A (S
EA),
Stap
hylo
cocc
al e
nter
otox
in D
(SED
), an
d Sh
iga
toxi
n.
Mos
t rem
arka
bly,
this
ant
i-SEB
sdA
b ha
d an
ext
rem
ely
high
Tm
of 8
5 de
gree
s C a
nd a
n ab
ility
to re
fold
aft
er h
eatin
g to
95
degr
ees
C. Th
e sh
arp
Tm d
eter
min
ed b
y ci
rcul
ar d
ichr
oism
, was
foun
d to
con
tras
t with
the
grad
ual d
ecre
ase
obse
rved
in in
trin
sic
fluor
es-
cenc
e. W
e de
mon
stra
ted
the
utili
ty o
f thi
s sdA
b as
a c
aptu
re a
nd d
etec
tor m
olec
ule
in L
umin
ex b
ased
ass
ays p
rovi
ding
lim
its o
f de
tect
ion
(LO
Ds)
of a
t lea
st 6
4 pg
/ml.
Con
clus
ion:
The
anti-
SEB
sdA
b A
3 w
as fo
und
to h
ave
a hi
gh a
ffini
ty a
nd a
n ex
trao
rdin
arily
hi
gh T
m a
nd c
ould
still
refo
ld to
reco
ver a
ctiv
ity a
fter
hea
t den
atur
atio
n. Th
is c
ombi
natio
n of
hea
t res
ilien
ce a
nd st
rong
, spe
cific
bi
ndin
g m
ake
this
sdA
b a
good
can
dida
te fo
r use
in a
ntib
ody-
base
d to
xin
dete
ctio
n te
chno
logi
es.
Gra
ef e
t al
. 201
1
Den
atur
ing
cond
itio
nsR
efol
ding
Ther
mod
ynam
ic st
abili
ty
A v
arie
ty o
f tec
hniq
ues,
incl
udin
g hi
gh-p
ress
ure
unfo
ldin
g m
onito
red
by F
ouri
er tr
ansf
orm
infr
ared
spec
tros
copy
, fluo
resc
ence
, ci
rcul
ar d
ichr
oism
, and
surf
ace
plas
mon
reso
nanc
e sp
ectr
osco
py, h
ave
been
use
d to
inve
stig
ate
the
equi
libri
um fo
ldin
g pr
oper
ties o
f si
x si
ngle
-dom
ain
antig
en b
inde
rs d
eriv
ed fr
om c
amel
id h
eavy
-cha
in a
ntib
odie
s with
spec
ifici
ties f
or ly
sozy
mes
, bet
a-la
ctam
ases
, and
a
dye
(RR6
). Va
riou
s den
atur
ing
cond
ition
s (gu
anid
iniu
m c
hlor
ide,
ure
a, te
mpe
ratu
re, a
nd p
ress
ure)
pro
vide
d co
mpl
emen
tary
and
in
depe
nden
t met
hods
for c
hara
cter
izin
g th
e st
abili
ty a
nd u
nfol
ding
pro
pert
ies o
f the
ant
ibod
y fr
agm
ents
. With
all
bind
ers,
com
plet
e re
cove
ry o
f the
bio
logi
cal a
ctiv
ity a
fter r
enat
urat
ion
dem
onst
rate
s tha
t che
mic
al- i
nduc
ed u
nfol
ding
is fu
lly re
vers
ible
. Fur
ther
mor
e,
dena
tura
tion
expe
rim
ents
follo
wed
by
optic
al sp
ectr
osco
pic
met
hods
and
affi
nity
mea
sure
men
ts in
dica
te th
at th
e an
tibod
y fr
agm
ents
ar
e un
fold
ed c
oope
rativ
ely
in a
sing
le tr
ansi
tion.
Thus
, unf
oldi
ng/r
efol
ding
equ
ilibr
ium
pro
ceed
s via
a si
mpl
e tw
o-st
ate
mec
hani
sm
(Nre
vers
ible
arr
ow U
), w
here
onl
y th
e na
tive
and
the
dena
ture
d st
ates
are
sign
ifica
ntly
pop
ulat
ed. Th
erm
ally
- ind
uced
den
atur
atio
n,
how
ever
, is n
ot c
ompl
etel
y re
vers
ible
, and
the
part
ial l
oss o
f bin
ding
cap
acity
mig
ht b
e du
e, a
t lea
st in
par
t, to
inco
rrec
t ref
oldi
ng
of th
e lo
ng lo
ops (
CD
Rs),
whi
ch a
re re
spon
sibl
e fo
r ant
igen
reco
gniti
on. M
ost i
nter
estin
gly,
all t
he fr
agm
ents
are
rath
er re
sist
ant t
o he
at-in
duce
d de
natu
ratio
n (a
ppar
ent T
-m =
60–
80 d
egre
es-C
), an
d di
spla
y hi
gh c
onfo
rmat
iona
l sta
bilit
ies (
Del
taG
(H2O
) = 3
0–60
kJ/
mol
). Su
ch h
igh
ther
mod
ynam
ic st
abili
ty h
as n
ever
bee
n re
port
ed fo
r any
func
tiona
l con
vent
iona
l ant
ibod
y fr
agm
ent,
even
whe
n en
gi-
neer
ed a
ntig
en b
inde
rs a
re c
onsi
dere
d. H
ence
, the
redu
ced
size
, im
prov
ed so
lubi
lity,
and
high
er st
abili
ty o
f the
cam
elid
hea
vy-c
hain
an
tibod
y fr
agm
ents
are
of s
peci
al in
tere
st fo
r bio
tech
nolo
gica
l and
med
ical
app
licat
ions
.
Dum
ou-
lin e
t al.
2002
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
470
Hea
t sho
ckC
onfo
rmat
ion
stud
ies
Den
atur
atio
nR
efol
ding
In a
pre
viou
s stu
dy w
e ha
ve sh
own
that
llam
a V
HH
ant
ibod
y fr
agm
ents
are
abl
e to
bin
d th
eir a
ntig
en a
fter
a h
eat s
hock
of 9
0 de
gree
s C, i
n co
ntra
st to
the
mur
ine
mon
oclo
nal a
ntib
odie
s. H
owev
er, t
he m
olec
ular
mec
hani
sm b
y w
hich
ant
ibod
y: a
ntig
en
inte
ract
ion
occu
rs u
nder
thes
e ex
trem
e co
nditi
ons r
emai
ns u
ncle
ar. T
o ex
amin
e in
mor
e de
tail
the
stru
ctur
al a
nd th
erm
odyn
amic
as
pect
s of t
he b
indi
ng m
echa
nism
, an
exte
nsiv
e C
D, I
TC, a
nd N
MR
stud
y w
as in
itiat
ed. I
n th
is st
udy
the
inte
ract
ion
betw
een
the
llam
a V
HH
-R2
frag
men
t and
its a
ntig
en, t
he d
ye R
eact
ive
Red-
6 (R
R6) h
as b
een
expl
ored
. The
data
show
cle
arly
that
mos
t of t
he
VH
H-R
2 po
pula
tion
at 8
0 de
gree
s C is
in a
n un
fold
ed c
onfo
rmat
ion.
In c
ontr
ast,
CD
spec
tra
repr
esen
ting
the
com
plex
bet
wee
n V
HH
-R2
and
the
dye
rem
aine
d th
e sa
me
up to
80
degr
ees C
. Int
eres
tingl
y, ad
ditio
n of
the
dye
to th
e de
natu
red
VH
H-R
2 at
80
degr
ees C
yie
lded
the
spec
trum
of t
he n
ativ
e co
mpl
ex. Th
ese
resu
lts su
gges
t an
indu
ced
refo
ldin
g of
den
atur
ed V
HH
-R2
by it
s an
tigen
und
er th
ese
extr
eme
cond
ition
s. Th
is in
duce
d re
fold
ing
show
ed so
me
sim
ilari
ties w
ith th
e w
ell e
stab
lishe
d “in
duce
d fit
” m
echa
nism
of a
ntib
ody-
antig
en in
tera
ctio
ns a
t am
bien
t tem
pera
ture
. How
ever
, the
mai
n di
ffere
nce
with
the
“indu
ced
fit01
48”
mec
hani
sm is
that
at t
he st
art o
f the
add
ition
of t
he a
ntig
en m
ost o
f the
VH
H m
olec
ules
are
in a
n un
fold
ed c
onfo
rmat
ion.
The
refo
ldin
g ca
pabi
lity
unde
r the
se e
xtre
me
cond
ition
s and
the
stab
le c
ompl
ex fo
rmat
ion
mak
e V
HH
s use
ful i
n a
wid
e va
riet
y of
app
li-ca
tions
.
Dol
k et
al
. 200
5b
Bac
illus
ant
hrac
isTh
erm
al s
tabi
lity
Den
atur
atio
nR
efol
ding
Sign
ifica
nt e
ffort
s to
deve
lop
both
labo
rato
ry a
nd fi
eld-
base
d de
tect
ion
assa
ys fo
r an
arra
y of
pot
entia
l bio
logi
cal t
hrea
ts st
arte
d w
ell b
efor
e th
e an
thra
x at
tack
s of 2
001
and
have
con
tinue
d w
ith re
new
ed u
rgen
cy fo
llow
ing.
Whi
le n
umer
ous a
ssay
s and
met
h-od
s hav
e be
en e
xplo
red
that
are
suita
ble
for l
abor
ator
y ut
iliza
tion,
det
ectio
n in
the
field
is o
ften
com
plic
ated
by
requ
irem
ents
for
func
tiona
lity
in a
uste
re e
nvir
onm
ents
, whe
re li
mite
d co
ld-c
hain
faci
litie
s exi
st. I
n an
effo
rt to
ove
rcom
e th
ese
assa
y lim
itatio
ns fo
r Ba
cillu
s ant
hrac
is, o
ne o
f the
mos
t rec
ogni
zabl
e th
reat
s, a
seri
es o
f sin
gle
dom
ain
antib
odie
s (sd
Abs
) wer
e is
olat
ed fr
om a
pha
ge
disp
lay
libra
ry p
repa
red
from
imm
uniz
ed ll
amas
. Cha
ract
eriz
atio
n of
targ
et sp
ecifi
city
, affi
nity
, and
ther
mal
stab
ility
was
con
duct
ed
for s
ix sd
Ab
fam
ilies
isol
ated
from
roun
ds o
f sel
ectio
n ag
ains
t the
bac
teri
al sp
ore.
The
prot
ein
targ
et fo
r all
six
sdA
b fa
mili
es w
as
dete
rmin
ed to
be
the
S-la
yer p
rote
in E
A1,
whi
ch is
pre
sent
in b
oth
vege
tativ
e ce
lls a
nd b
acte
rial
spor
es. A
ll of
the
sdA
bs e
xam
ined
ex
hibi
ted
a hi
gh d
egre
e of
spec
ifici
ty fo
r the
targ
et b
acte
rium
and
its s
pore
, with
affi
nitie
s in
the
nano
mol
ar ra
nge,
and
the
abili
ty
to re
fold
into
func
tiona
l ant
igen
-bin
ding
mol
ecul
es fo
llow
ing
seve
ral r
ound
s of t
herm
al d
enat
urat
ion
and
refo
ldin
g. Th
is re
sear
ch
dem
onst
rate
s the
cap
abili
ties o
f the
se sd
Abs
and
thei
r pot
entia
l for
inte
grat
ion
into
cur
rent
and
dev
elop
ing
assa
ys a
nd b
iose
nsor
s.
Wal
per
et a
l. 20
12
Dan
druff
Mal
asse
zia
furf
urSh
ampo
oD
enat
urin
g co
ndit
ions
Am
ino
acid
rep
lace
men
t
As p
art o
f res
earc
h ex
plor
ing
the
feas
ibili
ty o
f usi
ng a
ntib
ody
frag
men
ts to
inhi
bit t
he g
row
th o
f org
anis
ms i
mpl
icat
ed in
dan
druff
, w
e is
olat
ed a
ntib
ody
frag
men
ts th
at b
ind
to a
cel
l sur
face
pro
tein
of M
alas
sezi
a fu
rfur
in th
e pr
esen
ce o
f sha
mpo
o. W
e fo
und
that
ph
age
disp
lay
of ll
ama
sing
le-d
omai
n an
tibod
y fr
agm
ents
(VH
Hs)
can
be
exte
nded
to v
ery
hars
h co
nditi
ons,
such
as t
he p
rese
nce
of sh
ampo
o co
ntai
ning
non
ioni
c an
d an
ioni
c su
rfac
tant
s. W
e se
lect
ed se
vera
l VH
Hs t
hat b
ind
to th
e ce
ll w
all p
rote
in M
alf1
of M
. fu
rfur
, a fu
ngus
impl
icat
ed in
cau
sing
dan
druff
. In
addi
tion
to h
igh
stab
ility
in th
e pr
esen
ce o
f sha
mpo
o, th
ese
VH
Hs a
re a
lso
stab
le
unde
r oth
er d
enat
urin
g co
nditi
ons,
such
as h
igh
urea
con
cent
ratio
ns. M
any
of th
e st
able
VH
Hs w
ere
foun
d to
con
tain
arg
inin
e at
po
sitio
n 44
. Rep
lace
men
t of t
he n
ativ
e am
ino
acid
at p
ositi
on 4
4 w
ith a
rgin
ine
in th
e m
ost s
tabl
e V
HH
that
lack
ed th
is a
rgin
ine
resu
lted
in a
dra
mat
ic fu
rthe
r inc
reas
e in
the
stab
ility
. The
com
bina
tion
of th
e un
ique
pro
pert
ies o
f VH
Hs t
oget
her w
ith a
pplie
d ph
age
disp
lay
and
prot
ein
engi
neer
ing
is a
pow
erfu
l met
hod
for o
btai
ning
hig
hly
stab
le V
HH
s tha
t can
be
used
in a
wid
e ra
nge
of
appl
icat
ions
.
Dol
k et
al
. 200
5a
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
471
Solu
bilit
yC
DR
3H
ydro
phili
c am
ino
acid
sTr
ypan
osom
e su
rfac
e gl
ycop
rote
in
Hea
vy c
hain
onl
y an
tibod
ies o
f cam
elid
s bin
d th
eir a
ntig
ens w
ith a
sing
le d
omai
n, th
e V
HH
, whi
ch a
cqui
red
adap
tatio
ns re
lativ
e to
cl
assi
cal V
Hs t
o fu
nctio
n in
the
abse
nce
of a
VL
part
ner.
Add
ition
al C
DR
loop
con
form
atio
ns, o
utsi
de th
e ca
noni
cal l
oop
stru
ctur
es
of V
Hs,
bro
aden
the
repe
rtoi
re o
f the
ant
igen
-bin
ding
site
. The
com
bine
d eff
ects
of p
art o
f the
CD
R3 th
at fo
lds o
ver t
he “f
orm
er”
VL
bind
ing
site
and
fram
ewor
k-2
mut
atio
ns to
mor
e hy
drop
hilic
am
ino
acid
s, e
nhan
ce th
e so
lubi
lity
of V
HH
dom
ains
and
pre
vent
V
L pa
irin
g. c
AbA
n33,
a V
HH
dom
ain
spec
ific
for t
he c
arbo
hydr
ate
moi
ety
of th
e va
rian
t sur
face
gly
copr
otei
n of
tryp
anos
omes
, ha
s a sh
ort C
DR3
loop
that
doe
s not
cov
er th
e fo
rmer
VL
bind
ing
site
as w
ell a
s a V
H-s
peci
fic T
rp47
inst
ead
of th
e V
HH
-spe
cific
G
ly47
. Res
urfa
cing
its f
ram
ewor
k-2
regi
on (m
utat
ions
Tyr
37Va
l, G
lu44
Gly
and
Arg
45Le
u) to
mim
ic th
at o
f a h
uman
VH
rest
ores
th
e V
L bi
ndin
g ca
paci
ty. I
n so
lutio
n, th
e hu
man
ised
VH
H b
ehav
es a
s a so
lubl
e, m
onom
eric
ent
ity, a
lbei
t with
redu
ced
ther
mod
y-na
mic
stab
ility
and
affi
nity
for i
ts a
ntig
en. C
ompa
riso
n of
the
crys
tal s
truc
ture
s of c
AbA
n33
and
its h
uman
ised
der
ivat
ive
reve
als
ster
ic h
indr
ance
exe
rted
by
VH
H-s
peci
fic re
sidu
es T
yr37
and
Arg
45 th
at p
reve
nt th
e V
L do
mai
n pa
irin
g, w
here
as G
lu44
and
Arg
45
are
key
elem
ents
to a
void
inso
lubi
lity
of th
e do
mai
n.
Con
rath
et
al.
2005
Seru
m h
alf-
live
Porc
ine
im
mun
oglo
bulin
GE.
col
i F4
fimbr
iae
The
ther
apeu
tic p
aren
tera
l app
licat
ion
of ll
ama
sing
le-d
omai
n an
tibod
y fr
agm
ents
(VH
Hs)
is h
ampe
red
by th
eir
smal
l siz
e,
resu
lting
in a
fast
elim
inat
ion
from
the
body
. Her
e w
e de
scri
be a
met
hod
to in
crea
se th
e se
rum
hal
f-lif
e of
VH
Hs
in p
igs
by
fusi
on to
ano
ther
VH
H b
indi
ng to
por
cine
imm
unog
lobu
lin G
(pIg
G).
We
isol
ated
19
pIgG
-bin
ding
VH
Hs
from
an
imm
uniz
ed
llam
a us
ing
phag
e di
spla
y. S
ix V
HH
s w
ere
gene
tical
ly fu
sed
to m
odel
VH
H K
609
that
bin
ds to
Esc
heri
chia
col
i F4
fimbr
iae.
All
six
yeas
t-pr
oduc
ed g
enet
ic fu
sion
s of
two
VH
H d
omai
ns (V
HH
2s) w
ere
func
tiona
l in
ELIS
A a
nd b
ound
to p
IgG
with
hig
h af
fin-
ity (1
–33
nM).
Four
pIg
G-b
indi
ng V
HH
2s w
ere
adm
inis
tere
d to
pig
s an
d sh
owed
a 1
00-f
old
exte
nded
in v
ivo
resi
denc
e tim
es a
s co
mpa
red
to a
con
trol
VH
H2
that
doe
s no
t bin
d to
pIg
G. T
his
coul
d pr
ovid
e th
e ba
sis
for
ther
apeu
tic a
pplic
atio
n of
VH
Hs
in
pigs
.
Har
mse
n et
al.
2005
Bio
dist
ribu
tion
stu
dies
Blo
od c
ircu
lati
onTu
mou
r re
tent
ion
Tum
our-
targ
etin
g ve
hicl
e
Two
cam
el si
ngle
-dom
ain
frag
men
ts, c
Ab-
Lys2
and
cA
b-Ly
s3, r
ecog
nizi
ng a
n ov
erla
ppin
g ep
itope
of l
ysoz
yme
with
a d
isso
ciat
ion
cons
tant
of 2
nM
and
6S
nM, r
espe
ctiv
ely,
and
a bi
vale
nt c
Ab-
Lys3
wer
e in
vest
igat
ed fo
r the
ir a
bilit
y to
targ
et tr
ansg
enic
tum
ors
expr
essi
ng ly
sozy
me
on th
eir m
embr
ane.
Bio
dist
ribu
tion
stud
ies r
evea
led
that
thes
e no
n-im
mun
ogen
ic m
onom
eric
and
biv
alen
t ca
mel
sing
le-d
omai
n an
tigen
bin
ders
spec
ifica
lly ta
rget
lyso
zym
e-ex
pres
sing
tum
ors a
nd m
etas
tatic
lesi
ons.
The
exce
ss o
f ant
i-bo
dy is
rapi
dly
elim
inat
ed fr
om th
e bl
ood
circ
ulat
ion
and
no c
Ab
rete
ntio
n w
as o
bser
ved
in n
orm
al o
rgan
s. Th
e tu
mor
to o
rgan
cA
b-ra
tios a
t 2 a
nd 8
h w
ere
in th
e (2
.1–1
0.8)
: 1
and
(6.2
–23.
7) :
1 ra
nge,
resp
ectiv
ely.
The
degr
ee a
nd sp
ecifi
city
of t
umor
rete
n-tio
n is
inde
pend
ent o
f the
affi
nity
of t
he re
com
bina
nt c
amel
sing
le-d
omai
n fr
agm
ents
for t
heir
ant
igen
and
from
thei
r uni
vale
nt
mon
omer
ic (1
5 kD
a) o
r biv
alen
t for
mat
(33
kDa)
. This
stud
y de
mon
stra
tes t
he su
cces
sful
and
spec
ific
in v
ivo
targ
etin
g of
tum
ors
by c
amel
sing
le-d
omai
n fr
agm
ents
. It m
ay o
pen
pers
pect
ives
for t
heir
futu
re u
se a
s tum
or-t
arge
ting
vehi
cle,
due
to th
eir s
mal
l si
ze, s
olub
le b
ehav
iour
and
bec
ause
they
are
non
-imm
unog
enic
and
inte
ract
with
epi
tope
s tha
t are
less
ant
igen
ic fo
r con
vent
iona
l an
tibod
ies.
Cor
tez-
Reta
-m
ozo
et
al. 2
002
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
472
Bin
ding
kin
etic
sR
icin
toxo
idEL
ISA
Surf
ace
plas
mon
res
o-na
nce
Sing
le d
omai
n an
tibod
ies a
re th
e re
com
bina
ntly
exp
ress
ed b
indi
ng fr
agm
ents
der
ived
from
hea
vy c
hain
ant
ibod
ies f
ound
in c
amel
s an
d lla
mas
. Thes
e un
ique
bin
ding
ele
men
ts o
ffer m
any
desi
rabl
e pr
oper
ties s
uch
as th
eir s
mal
l siz
e (s
imila
r to
15 k
Da)
and
ther
-m
al st
abili
ty, w
hich
mak
es th
em a
ttra
ctiv
e al
tern
ativ
es to
con
vent
iona
l mon
oclo
nal a
ntib
odie
s. W
e cr
eate
d a
phag
e di
spla
y lib
rary
fr
om ll
amas
imm
uniz
ed w
ith ri
cin
toxo
id a
nd se
lect
ed a
num
ber o
f sin
gle
dom
ain
antib
odie
s. P
hage
sele
cted
on
rici
n w
ere
foun
d to
bin
d to
eith
er ri
cin
A c
hain
or t
he in
tact
mol
ecul
e; n
o ri
cin
B ch
ain
bind
ers w
ere
iden
tified
. By
pann
ing
on B
cha
in, w
e id
entifi
ed
bind
ers a
nd h
ave
char
acte
rize
d th
eir b
indi
ng to
the
rici
n B
chai
n. W
hile
they
hav
e a
poor
er a
ffini
ty th
an th
e pr
evio
usly
des
crib
ed A
ch
ain
bind
ers,
it w
as fo
und
that
they
per
form
ed d
ram
atic
ally
bet
ter a
s cap
ture
reag
ents
for t
he d
etec
tion
of ri
cin,
pro
vidi
ng a
lim
it of
det
ectio
n in
enz
yme
linke
d im
mun
osor
bent
ass
ay (E
LISA
) bel
ow 1
00 p
g/m
L an
d ex
celle
nt sp
ecifi
city
for r
icin
ver
sus t
he h
ighl
y re
late
d RC
A 1
20 (1
to 1
0 00
0). W
e al
so re
eval
uate
d th
e pr
evio
usly
isol
ated
ant
iric
in si
ngle
dom
ain
antib
ody
bind
ing
kine
tics u
sing
su
rfac
e pl
asm
on re
sona
nce
and
foun
d th
eir K
(d)s
mat
ched
clo
sely
to th
ose
prev
ious
ly o
btai
ned
unde
r equ
ilibr
ium
bin
ding
con
di-
tions
mea
sure
d us
ing
the
Lum
inex
flow
cyt
omet
er.
And
er-
son
et a
l. 20
10
Nan
obod
y pr
oper
ties
Epid
erm
al g
row
th fa
ctor
re
cept
orD
rug
targ
etin
g
The
dis
cove
ry o
f nat
ural
ly o
ccur
ring
hea
vy c
hain
onl
y an
tibo
dies
and
thei
r fu
rthe
r de
velo
pmen
t int
o sm
all r
ecom
bina
nt
‘nan
obod
ies’
offe
rs a
ttra
ctiv
e ap
plic
atio
ns in
dru
g ta
rget
ing.
Her
e, w
e de
scri
be th
e pr
oper
ties
of n
anob
odie
s th
at h
ave
been
de
velo
ped
to ta
rget
the
epid
erm
al g
row
th fa
ctor
rec
epto
r (E
GFR
) and
con
tras
t the
se to
the
char
acte
rist
ics
of h
eavy
cha
in o
nly
anti
bodi
es a
nd c
onve
ntio
nal a
ntib
odie
s. E
GFR
is o
vere
xpre
ssed
in m
any
tum
ors
and
is a
n at
trac
tive
targ
et fo
r tu
mor
-dir
ecte
d dr
ug ta
rget
ing.
Alti
ntas
et
al.
2012
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
473
Tabl
e 6A
. App
licat
ion
of s
ingl
e-do
mai
n an
tibo
dy fr
agm
ents
in th
erap
y: in
hibi
tion
of e
nzym
es, t
oxin
s an
d ot
her
solu
ble
prot
eins
Prot
ein
kina
se C
Enzy
me
mod
ulat
orTh
e 10
isoz
ymes
of t
he p
rote
in k
inas
e C
(PK
C) f
amily
can
hav
e di
ffere
nt ro
les o
n th
e sa
me
biol
ogic
al p
roce
ss, m
akin
g is
ozym
e sp
ecifi
c an
alys
is o
f fun
ctio
n cr
ucia
l. C
urre
ntly
, onl
y fe
w p
harm
acol
ogic
al c
ompo
unds
with
mod
erat
e is
ozym
e sp
ecifi
c eff
ects
exi
st
thus
ham
peri
ng re
sear
ch in
to in
divi
dual
PK
C is
ozym
es. Th
e an
tigen
bin
ding
regi
ons o
f cam
elid
sing
le c
hain
ant
ibod
ies (
VH
Hs)
co
uld
prov
ide
a so
lutio
n fo
r obt
aini
ng P
KC
isoz
yme
spec
ific
mod
ulat
ors.
In th
e pr
esen
t stu
dy, w
e ha
ve su
cces
sful
ly se
lect
ed a
nd
char
acte
rize
d PK
C e
psilo
n sp
ecifi
c V
HH
ant
ibod
ies f
rom
two
imm
une
VH
H li
brar
ies u
sing
pha
ge d
ispl
ay. Th
e V
HH
s wer
e sh
own
to e
xclu
sive
ly b
ind
to P
KC
eps
ilon
in E
LISA
and
imm
unop
reci
pita
tion
stud
ies.
Str
ikin
gly,
five
of th
e V
HH
s had
an
effec
t on
PKC
ep
silo
n ki
nase
act
ivity
in v
itro.
VH
Hs A
10, C
1 an
d D
1 in
crea
sed
PKC
eps
ilon
kina
se a
ctiv
ity in
a c
once
ntra
tion-
depe
nden
t man
ner
(EC
(50)
val
ues:
212–
310
nM),
whe
reas
E6
and
G8
inhi
bite
d PK
C e
psilo
n ac
tivity
(IC
(50)
val
ues:
103–
233
nM).
Non
e of
thes
e V
HH
s had
an
effec
t on
the
activ
ity o
f the
oth
er n
ovel
PK
C is
ozym
es P
KC
del
ta a
nd P
KC
thet
a. T
o ou
r kno
wle
dge,
thes
e an
tibod
ies
are
the
first
des
crib
ed V
HH
act
ivat
ors a
nd in
hibi
tors
for a
pro
tein
kin
ase.
Fur
ther
mor
e, th
e de
velo
pmen
t of P
KC
eps
ilon
spec
ific
mod
ulat
ors i
s an
impo
rtan
t con
trib
utio
n to
PK
C re
sear
ch.
Paal
anen
et
al.
2011
Ant
i-id
ioty
pic
Alli
inas
eM
olec
ular
mim
icry
Abz
yme
Scre
enin
g of
inhi
bito
ry A
b1 a
ntib
odie
s is
a c
ritic
al s
tep
for
prod
ucin
g ca
taly
tic a
ntib
odie
s in
the
anti-
idio
typi
c ap
proa
ch. H
ow-
ever
, the
inco
mpa
tible
sur
face
of t
he a
ctiv
e si
te o
f the
enz
yme
and
the
antig
en-b
indi
ng s
ite o
f het
erot
etra
mer
ic c
onve
ntio
nal
antib
odie
s be
com
e th
e lim
iting
ste
p. B
ecau
se c
amel
id-d
eriv
ed n
anob
odie
s po
sses
s th
e po
tent
ial t
o pr
efer
entia
lly b
ind
to th
e ac
tive
site
of e
nzym
es d
ue to
thei
r sm
all s
ize
and
long
CD
R3, w
e ha
ve d
evel
oped
a n
ovel
app
roac
h to
pro
duce
ant
ibod
ies
with
al
liina
se a
ctiv
ities
by
expl
oitin
g th
e m
olec
ular
mim
icry
of c
amel
nan
obod
ies.
By
scre
enin
g th
e ca
mel
id-d
eriv
ed v
aria
ble
regi
on
of th
e he
avy
chai
n cD
NA
pha
ge d
ispl
ay li
brar
y w
ith a
lliin
ase,
we
obta
ined
an
inhi
bito
ry n
anob
ody
VH
HA
4 th
at re
cogn
izes
the
activ
e si
te. F
urth
er s
cree
ning
with
VH
HA
4 fr
om th
e sa
me
vari
able
dom
ain
of th
e he
avy
chai
n of
a h
eavy
-cha
in a
ntib
ody
libra
ry
led
to a
hig
her
inci
denc
e of
ant
i-id
ioty
pic
Ab2
abz
ymes
with
alli
inas
e ac
tiviti
es. O
ne o
f the
abz
ymes
, VH
HC
10, s
how
ed th
e hi
ghes
t act
ivity
that
can
be
inhi
bite
d by
Ab1
VH
HA
4 an
d al
liina
se c
ompe
titiv
e in
hibi
tor
peni
cilla
min
e an
d si
gnifi
cant
ly s
up-
pres
sed
the
B16
tum
or c
ell g
row
th in
the
pres
ence
of a
lliin
in v
itro.
The
resu
lts h
ighl
ight
the
feas
ibili
ty o
f pro
duci
ng a
bzym
es
via
anti-
idio
typi
c na
nobo
dy a
ppro
ach.
Li e
t al.
2012
Allo
ster
ic e
ffect
orD
ihyd
rofo
late
red
ucta
seIn
hibi
tion
kin
etic
sC
ryst
al s
truc
ture
Alth
ough
it h
as b
een
know
n fo
r man
y ye
ars t
hat a
ntib
odie
s dis
play
pro
pert
ies c
hara
cter
istic
of a
llost
eric
effe
ctor
s, th
e m
olec
ular
m
echa
nism
s res
pons
ible
for t
hese
effe
cts r
emai
n po
orly
und
erst
ood.
Her
e, w
e de
scri
be a
sing
le-d
omai
n an
tibod
y fr
agm
ent (
nano
-bo
dy) t
hat m
odul
ates
pro
tein
func
tion
by c
onst
rain
ing
conf
orm
atio
nal c
hang
e in
the
enzy
me
dihy
drof
olat
e re
duct
ase
(DH
FR).
Nan
obod
y 21
6 (N
b216
) beh
aves
as a
pot
ent a
llost
eric
inhi
bito
r of D
HFR
, giv
ing
rise
to m
ixed
hyp
erbo
lic in
hibi
tion
kine
tics.
The
crys
tal s
truc
ture
of N
b216
in c
ompl
ex w
ith D
HFR
reve
als t
hat t
he n
anob
ody
bind
s adj
acen
t to
the
activ
e si
te. H
alf o
f the
epi
tope
co
nsis
ts o
f res
idue
s fro
m th
e fle
xibl
e M
et20
loop
. This
loop
, whi
ch o
rdin
arily
osc
illat
es b
etw
een
occl
uded
and
clo
sed
conf
orm
a-tio
ns d
urin
g ca
taly
sis,
ass
umes
the
occl
uded
con
form
atio
n in
the
Nb2
16-b
ound
stat
e. U
sing
stop
ped
flow
, we
show
that
Nb2
16
inhi
bits
DH
FR b
y st
abili
sing
the
occl
uded
Met
20 lo
op c
onfo
rmat
ion.
Sur
pris
ingl
y, ki
netic
dat
a in
dica
te th
at th
e M
et20
loop
reta
ins
suffi
cien
t con
form
atio
nal fl
exib
ility
in th
e N
b216
-bou
nd st
ate
to a
llow
slow
subs
trat
e tu
rnov
er to
occ
ur.
Oye
n et
al
. 201
1
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
474
Sial
idas
eTr
ypan
osom
a cr
uzi
The
sia
lic a
cid
pres
ent i
n th
e pr
otec
tive
surf
ace
muc
in c
oat o
f Try
pano
som
a cr
uzi i
s ad
ded
by a
mem
bran
e an
chor
ed tr
ans-
sial
idas
e (T
cTS)
, a m
odifi
ed s
ialid
ase
that
is e
xpre
ssed
from
a la
rge
gene
fam
ily. I
n th
is w
ork,
we
anal
yzed
sin
gle
dom
ain
cam
elid
an
tibod
ies
prod
uced
aga
inst
tran
s-si
alid
ase.
Lla
mas
wer
e im
mun
ized
with
a re
com
bina
nt tr
ans-
sial
idas
e an
d in
hibi
tory
sin
gle-
dom
ain
antib
ody
frag
men
ts w
ere
obta
ined
by
phag
e di
spla
y se
lect
ion,
taki
ng a
dvan
tage
of a
scr
eeni
ng s
trat
egy
usin
g an
inhi
bi-
tion
test
inst
ead
of th
e cl
assi
c bi
ndin
g as
say.
Fou
r si
ngle
dom
ain
antib
odie
s di
spla
ying
str
ong
tran
s-si
alid
ase
inhi
bitio
n ac
tivity
ag
ains
t the
reco
mbi
nant
enz
yme
wer
e id
entif
ied.
The
y sh
are
the
sam
e co
mpl
emen
tari
ty-d
eter
min
ing
regi
on 3
leng
th (1
7 re
si-
dues
) and
hav
e ve
ry s
imila
r se
quen
ces.
Thi
s re
sult
indi
cate
s th
at th
ey li
kely
der
ived
from
a u
niqu
e cl
one.
Pro
babl
y th
ere
is o
nly
one
stru
ctur
al s
olut
ion
for
tight
bin
ding
inhi
bito
ry a
ntib
odie
s ag
ains
t the
TcT
S us
ed fo
r im
mun
izat
ion.
To
our
surp
rise
, thi
s si
ngle
dom
ain
antib
ody
that
inhi
bits
the
reco
mbi
nant
TcT
S, fa
iled
to in
hibi
t the
enz
ymat
ic a
ctiv
ity p
rese
nt in
par
asite
ext
ract
s.
Ana
lysi
s of
indi
vidu
al re
com
bina
nt tr
ans-
sial
idas
es s
how
ed th
at e
nzym
es e
xpre
ssed
from
diff
eren
t gen
es w
ere
inhi
bite
d to
dif-
fere
nt e
xten
ts (f
rom
8 to
98%
) by
the
llam
a an
tibod
ies.
Am
ino
acid
cha
nges
at k
ey p
ositi
ons
are
likel
y to
be
resp
onsi
ble
for
the
diffe
renc
es in
inhi
bitio
n fo
und
amon
g th
e re
com
bina
nt e
nzym
es. T
hese
resu
lts s
ugge
st th
at th
e pr
esen
ce o
f a la
rge
and
dive
rse
tran
s-si
alid
ase
fam
ily m
ight
be
requ
ired
to p
reve
nt th
e in
hibi
tory
resp
onse
aga
inst
this
ess
entia
l enz
yme
and
mig
ht th
us c
onst
i-tu
te a
nov
el s
trat
egy
of T
. cru
zi to
eva
de th
e ho
st im
mun
e sy
stem
.
Ratie
r et
al. 2
008
Clo
stri
dium
diffi
cile
Nos
ocom
ial i
nfec
tion
Nor
th A
mer
ica
Exot
oxin
A/B
Gas
troi
ntes
tina
l tra
ct
Clo
stri
dium
diff
icile
is a
lead
ing
caus
e of
nos
ocom
ial i
nfec
tion
in N
orth
Am
eric
a an
d a
cons
ider
able
cha
lleng
e to
hea
lthca
re
prof
essi
onal
s in
hos
pita
ls a
nd n
ursi
ng h
omes
. The
Gra
m-p
ositi
ve b
acte
rium
pro
duce
s tw
o hi
gh m
olec
ular
wei
ght e
xoto
xins
, to
xin
A (T
cdA
) and
toxi
n B
(Tcd
B), w
hich
are
the
maj
or v
irul
ence
fact
ors
resp
onsi
ble
for
C. d
iffic
ile-a
ssoc
iate
d di
seas
e an
d ar
e ta
rget
s fo
r C
. diff
icile
-ass
ocia
ted
dise
ase
ther
apy.
Her
e, re
com
bina
nt s
ingl
e-do
mai
n an
tibod
y fr
agm
ents
(V(H
)Hs)
, whi
ch
spec
ifica
lly ta
rget
the
cell
rece
ptor
bin
ding
dom
ains
of T
cdA
or
TcdB
, wer
e is
olat
ed fr
om a
n im
mun
e lla
ma
phag
e di
spla
y lib
rary
an
d ch
arac
teri
zed.
Fou
r V
(H)H
s (A
4.2,
A5.
1, A
20.1
, and
A26
.8),
all s
how
n to
reco
gniz
e co
nfor
mat
iona
l epi
tope
s, w
ere
pote
nt
neut
raliz
ers
of th
e cy
topa
thic
effe
cts
of to
xin
A o
n fib
robl
ast c
ells
in a
n in
vitr
o as
say.
The
neu
tral
izin
g po
tenc
y w
as fu
rthe
r en
hanc
ed w
hen
V(H
)Hs
wer
e ad
min
iste
red
in p
aire
d or
trip
let c
ombi
natio
ns a
t the
sam
e ov
eral
l V(H
)H c
once
ntra
tion,
sug
gest
-in
g re
cogn
ition
of n
onov
erla
ppin
g Tc
dA e
pito
pes.
Bia
core
epi
tope
map
ping
exp
erim
ents
reve
aled
that
som
e sy
nerg
istic
com
-bi
natio
ns c
onsi
sted
of V
(H)H
s re
cogn
izin
g ov
erla
ppin
g ep
itope
s, a
n in
dica
tion
that
fact
ors
othe
r th
an m
ere
epito
pe b
lock
ing
are
resp
onsi
ble
for
the
incr
ease
d ne
utra
lizat
ion.
Fur
ther
bin
ding
ass
ays
reve
aled
Tcd
A-s
peci
fic V
(H)H
s ne
utra
lized
toxi
n A
by
bind
ing
to s
ites
othe
r th
an th
e ca
rboh
ydra
te b
indi
ng p
ocke
t of t
he to
xin.
With
favo
rabl
e ch
arac
teri
stic
s su
ch a
s hi
gh p
rodu
ctio
n yi
eld,
pot
ent t
oxin
neu
tral
izat
ion,
and
intr
insi
c st
abili
ty, t
hese
V(H
)Hs
are
attr
activ
e sy
stem
ic th
erap
eutic
s bu
t are
mor
e so
as
oral
ther
apeu
tics
in th
e de
stab
ilizi
ng e
nvir
onm
ent o
f the
gas
troi
ntes
tinal
trac
t.
Hus
sack
et
al.
2011
b
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
475
Stap
hylo
cocc
us a
ureu
sH
emol
ysin
‘Hot
-col
d’ h
emol
ytic
ac
tivi
ty
Obj
ectiv
e: T
o is
olat
e an
d ch
arac
teri
ze S
taph
yloc
occu
s aur
eus (
S. a
ureu
s) h
emol
ysin
neu
tral
izin
g dA
bs fr
ont p
hage
dis
play
libr
ary
of In
dian
des
ert c
amel
. Met
hods
: Pha
ge d
ispl
ay li
brar
y of
5 ×
107 d
Ab
clon
es o
f LPS
-im
mun
ized
Indi
an d
eser
t cam
el c
onst
ruct
ed
in o
ur la
bora
tory
was
use
d fo
r sel
ectio
n of
S. a
ureu
s exo
toxi
n sp
ecifi
c cl
ones
by
pann
ing
tech
niqu
e. E
nric
hmen
t of A
g-sp
ecifi
c cl
ones
in su
cces
sive
roun
ds o
f pan
ning
was
ass
esse
d by
pha
ge-E
LISA
and
pha
ge ti
trat
ion.
Diff
eren
t dA
b cl
ones
bin
ding
to S
. aur
eus
exot
oxin
Ags
wer
e ex
pres
sed
with
C-t
erm
inal
6 X
His
tag
in E
. col
i and
pur
ified
by
Ni-
chel
ate
chro
mat
ogra
phy.
The
expr
essi
on
was
ver
ified
by
SDS-
PAG
E an
d w
este
rn b
lott
ing.
The
puri
fied
clon
es w
ere
test
ed fo
r inh
ibiti
on o
f ‘ho
t-co
ld’ h
emol
ytic
act
ivity
in
vitr
o. R
esis
tanc
e to
ther
mal
inac
tivat
ion
of th
e dA
b cl
ones
was
stud
ied
by o
bser
ving
the
effec
t of h
eat t
reat
men
t fro
m 5
0 de
gree
s C
to 9
9 de
gree
s C fo
r 30
min
on
the
‘hot
-col
d’ h
emol
ytic
act
ivity
in v
itro.
Res
ults
: Sev
eral
dA
b cl
ones
bin
ding
to S
. aur
eus e
xoto
xins
w
ere
isol
ated
and
enr
iche
d by
thre
e ro
unds
of p
anni
ng. Th
e so
lubl
e dA
b cl
ones
wer
e ap
prox
imat
ely
sim
ilar t
o 16
kD
a in
size
and
re
acte
d w
ith 6
X H
is ta
g sp
ecifi
c m
urin
e m
onoc
lona
l ant
ibod
y in
wes
tern
blo
t. O
ne o
f the
Ni-
chel
ate
affini
ty p
urifi
ed d
Ab.
6 X
His
cl
ones
, inh
ibite
d S.
aur
eus b
eta-
hem
olys
in a
ctiv
ity in
vitr
o an
d re
sist
ed th
erm
al in
activ
atio
n up
to 9
9 de
gree
s C. C
oncl
usio
ns: A
n S.
au
reus
bet
a-he
mol
ysin
neu
tral
izin
g dA
b cl
one
of p
ossi
ble
ther
apeu
tic p
oten
tial h
as b
een
isol
ated
.
Pooj
a an
d A
jit
2010
Thro
mbi
n ac
tiva
tabl
e fib
rino
lysi
s in
hibi
tor
Clo
t lys
is
Back
grou
nd: B
ecau
se a
ctiv
ated
thro
mbi
n ac
tivat
able
fibr
inol
ysis
inhi
bito
r (TA
FIa)
has
ver
y po
wer
ful a
ntifi
brin
olyt
ic p
rope
rtie
s, co
-ad
min
istra
tion
of t-
PA a
nd a
TA
FIa
inhi
bito
r enh
ance
s t-P
A tr
eatm
ent.
Obj
ectiv
e: W
e ai
med
to g
ener
ate
nano
bodi
es sp
ecifi
cally
inhi
bit-
ing
the
TAFI
a ac
tivity
and
to te
st th
eir e
ffect
on
t-PA
indu
ced
clot
lysis
. Res
ults
: Fiv
e na
nobo
dies
, rai
sed
tow
ards
an
activ
ated
mor
e st
able
TA
FIa
mut
ant (
TAFI
a A
(147
)-C
(305
)-I(3
25)-
I(329
)-Y(3
33)-
Q(3
35)),
are
des
crib
ed. Th
ese
nano
bodi
es in
hibi
t spe
cific
ally
TA
FIa
activ
ity,
resu
lting
in a
n in
hibi
tion
of u
p to
99%
at a
16-
fold
mol
ar e
xces
s of n
anob
ody
over
TA
FIa,
IC(5
0)’s
rang
e be
twee
n 0.
38- a
nd >
16-
fold
m
olar
exc
ess.
In v
itro
clot
lysis
exp
erim
ents
in th
e ab
senc
e of
thro
mbo
mod
ulin
(TM
) dem
onst
rate
that
the
nano
bodi
es e
xhib
it pr
ofi-
brin
olyt
ic e
ffect
s. H
owev
er, i
n th
e pr
esen
ce o
f TM
, one
nan
obod
y ex
hibi
ts a
n an
tifibr
inol
ytic
effe
ct w
here
as th
e ot
her n
anob
odie
s sho
w a
sli
ght a
ntifi
brin
olyt
ic e
ffect
at l
ow c
once
ntra
tions
and
a p
rono
unce
d pr
ofibr
inol
ytic
effe
ct a
t hig
her c
once
ntra
tions
. This
biph
asic
pat
tern
w
as h
ighl
y de
pend
ent o
n TM
and
t-PA
con
cent
ratio
n. Th
e na
nobo
dies
wer
e fo
und
to b
ind
in th
e ac
tive-
site
regi
on o
f TA
FIa
and
thei
r tim
e-de
pend
ent d
iffer
entia
l bin
ding
beh
avio
r dur
ing
TAFI
a in
activ
atio
n re
veal
ed th
e oc
curr
ence
of a
yet
unk
now
n in
term
edia
te c
onfo
r-m
atio
nal t
rans
ition
. Con
clus
ion:
Thes
e na
nobo
dies
are
ver
y po
tent
TA
FIa
inhi
bito
rs a
nd c
onst
itute
use
ful t
ools
to a
ccel
erat
e fib
rinol
ysis
. O
ur d
ata
also
dem
onst
rate
that
the
profi
brin
olyt
ic e
ffect
of T
AFI
a in
hibi
tion
may
be
reve
rsed
by
the
pres
ence
of T
M. Th
e id
entifi
catio
n of
a
new
con
form
atio
nal t
rans
ition
pro
vide
s new
insig
hts i
nto
the
conf
orm
atio
nal i
nact
ivat
ion
of th
e un
stab
le T
AFI
a.
Hen
-dr
ickx
et
al. 2
011
Tum
our
necr
osis
fact
orFu
sion
Elas
tin-
like
poly
pept
ide
Toba
cco
Tum
our n
ecro
sis f
acto
r (T
NF)
is a
maj
or p
ro-in
flam
mat
ory
cyto
kine
invo
lved
in m
ultip
le in
flam
mat
ory
dise
ases
. The
detr
imen
tal
activ
ity o
f TN
F ca
n be
blo
cked
by
vari
ous a
ntag
onis
ts, a
nd c
omm
erci
al th
erap
eutic
s bas
ed u
pon
this
pri
ncip
le h
ave
been
app
rove
d fo
r tre
atm
ent o
f dis
ease
s inc
ludi
ng rh
eum
atoi
d ar
thri
tis, C
rohn
’s di
seas
e an
d ps
oria
sis.
In a
sear
ch fo
r new
, im
prov
ed a
nti-
infla
mm
ator
y th
erap
eutic
s we
have
des
igne
d a
sing
le-d
omai
n m
onoc
lona
l ant
ibod
y (V
(H)H
), w
hich
reco
gniz
es T
NF.
The
antib
ody
com
pone
nt (T
NF-
V(H
)H) i
s bas
ed u
pon
an a
nti-h
uman
TN
F C
amel
idae
hea
vy-c
hain
mon
oclo
nal a
ntib
ody,
whi
ch w
as li
nked
to a
n el
astin
-like
pol
ypep
tide
(ELP
). W
e de
mon
stra
te th
at E
LP fu
sion
to th
e T
NF-
V(H
)H e
nhan
ces a
ccum
ulat
ion
of th
e fu
sion
pro
tein
du
ring
bio
man
ufac
turi
ng in
tran
sgen
ic to
bacc
o pl
ants
. With
this
stud
y, w
e sh
ow fo
r the
firs
t tim
e th
at th
is p
lant
-der
ived
ant
i-hu
man
TN
F-V
(H)H
ant
ibod
y w
as b
iolo
gica
lly a
ctiv
e in
viv
o. Th
eref
ore,
ther
apeu
tic a
pplic
atio
n of
TN
F-V
(H)H
-ELP
fusi
on p
rote
in
was
test
ed in
hum
aniz
ed T
NF
mic
e an
d w
as sh
own
to b
e eff
ectiv
e in
pre
vent
ing
deat
h ca
used
by
sept
ic sh
ock.
The
in v
ivo
pers
is-
tenc
e of
the
ELPy
late
d an
tibod
y w
as si
mila
r to
24 fo
ld lo
nger
than
that
of n
on-E
LPyl
ated
TN
F-V
(H)H
.
Con
rad
et a
l. 20
11
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
476
L-pl
asti
nM
alig
nanc
yF-
acti
n bu
ndlin
gFi
lopo
dia
L-pl
astin
, a c
onse
rved
mod
ular
F-a
ctin
bun
dlin
g pr
otei
n, is
ect
opic
ally
exp
ress
ed in
tum
or c
ells
and
con
trib
utes
to c
ell m
alig
nanc
y an
d in
vasi
on. Th
e un
derl
ying
mol
ecul
ar m
echa
nism
s inv
olve
d re
mai
n un
clea
r, in
par
t, be
caus
e sp
ecifi
c in
hibi
tors
of L
-pla
stin
are
la
ckin
g. W
e us
ed re
com
bina
nt a
lpac
a-de
rive
d L-
plas
tin si
ngle
-dom
ain
antib
odie
s (na
nobo
dies
) as e
ffect
or o
f L-p
last
in fu
nctio
n in
cel
ls. K
ey fi
ndin
gs w
ere
com
pare
d w
ith L
-pla
stin
dow
n-re
gula
tion
by R
NA
i. W
e sh
ow th
at n
anob
odie
s str
ongl
y in
tera
ct w
ith
L-pl
astin
by
targ
etin
g di
scre
te c
onfo
rmat
iona
l epi
tope
s with
nan
omol
ar a
ffini
ty. A
nan
obod
y th
at se
lect
ivel
y in
tera
cts w
ith th
e ta
ndem
ABD
s in
L-pl
astin
com
plet
ely
inhi
bits
F-a
ctin
bun
dlin
g at
equ
imol
ar ra
tios,
in c
ontr
ast t
o a
cont
rol g
reen
fluo
resc
ent p
ro-
tein
(GFP
) nan
obod
y. Th
is “k
nock
out”
nan
obod
y in
hibi
ts fi
lopo
dia
form
atio
n, m
otili
ty, a
nd in
vasi
on w
hen
expr
esse
d in
PC
-3 c
ells
. L-
plas
tin R
NA
inte
rfer
ence
show
ed n
o si
gnifi
cant
effe
ct o
n fil
opod
ial i
nteg
rity
and
onl
y m
argi
nally
rest
rain
ed th
e m
otile
pro
pert
ies
of c
ells
. L-p
last
in n
anob
odie
s uni
quel
y ex
pose
a fu
ndam
enta
l rol
e fo
r thi
s pro
tein
in fi
lopo
dia
form
atio
n an
d ce
ll m
igra
tion.
Ther
e-fo
re, t
hese
mol
ecul
es re
pres
ent a
pot
ent i
nstr
umen
t to
abla
te fu
nctio
ns o
f str
uctu
ral p
rote
ins w
ithou
t man
ipul
atin
g ge
ne e
xpre
s-si
on. I
n ad
ditio
n, w
e sh
ow th
at th
ey c
an b
e in
stru
men
tal i
n un
cove
ring
new
func
tions
of p
rote
ins t
hat r
emai
n ob
scur
ed b
y RN
Ai.
Del
anot
e et
al.
2010
Tabl
e 6B
. App
licat
ion
of s
ingl
e-do
mai
n an
tibo
dy fr
agm
ents
in th
erap
y: a
ctiv
ity
mod
ulat
ion
of c
ell s
urfa
ce p
rote
ins
Nan
obod
y pr
oper
ties
Epid
erm
al g
row
th fa
ctor
re
cept
orD
rug
targ
etin
g
The
disc
over
y of
nat
ural
ly o
ccur
ring
hea
vy c
hain
onl
y an
tibod
ies a
nd th
eir f
urth
er d
evel
opm
ent i
nto
smal
l rec
ombi
nant
‘nan
obod
-ie
s’ off
ers a
ttra
ctiv
e ap
plic
atio
ns in
dru
g ta
rget
ing.
Her
e, w
e de
scri
be th
e pr
oper
ties o
f nan
obod
ies t
hat h
ave
been
dev
elop
ed to
ta
rget
the
epid
erm
al g
row
th fa
ctor
rece
ptor
(EG
FR) a
nd c
ontr
ast t
hese
to th
e ch
arac
teri
stic
s of h
eavy
cha
in o
nly
antib
odie
s and
co
nven
tiona
l ant
ibod
ies.
EG
FR is
ove
rexp
ress
ed in
man
y tu
mor
s and
is a
n at
trac
tive
targ
et fo
r tum
or-d
irec
ted
drug
targ
etin
g.
Alti
ntas
et
al.
2012
Vasc
ular
end
othe
lial
grow
th fa
ctor
re
cept
or-2
Neo
vasc
ular
izat
ion
Met
asta
sis
Vasc
ular
end
othe
lial g
row
th fa
ctor
rece
ptor
-2 (V
EGFR
2) is
an
impo
rtan
t tum
or-a
ssoc
iate
d re
cept
or a
nd b
lock
ade
of th
e V
EGF
rece
p-to
r sig
nalin
g ca
n le
ad to
the
inhi
bitio
n of
neo
vasc
ular
izat
ion
and
tum
or m
etas
tasis
. Nan
obod
ies a
re th
e sm
alle
st in
tact
ant
igen
bin
ding
fr
agm
ents
der
ived
from
hea
vy c
hain
-onl
y an
tibod
ies o
ccur
ring
in c
amel
ids.
Her
e, w
e de
scrib
e th
e id
entifi
catio
n of
a V
EGFR
2-sp
ecifi
c N
anob
ody,
nam
ed 3
VG
R19,
from
dro
med
arie
s im
mun
ized
with
a c
ell l
ine
expr
essin
g hi
gh le
vels
of V
EGFR
2. W
e de
mon
stra
te b
y FA
CS,
th
at 3
VG
R19
Nan
obod
y sp
ecifi
cally
bin
ds V
EGFR
2 on
the
surf
ace
of 2
93K
DR
and
HU
VEC
s cel
ls. F
urth
erm
ore,
the
3VG
R19
Nan
obod
y po
tent
ly in
hibi
ts fo
rmat
ion
of c
apill
ary-
like
stru
ctur
es. Th
ese
data
show
the
pote
ntia
l of N
anob
odie
s for
the
bloc
kade
of V
EGFR
2 sig
nal-
ing
and
prov
ide
a ba
sis fo
r the
dev
elop
men
t of n
ovel
can
cer t
hera
peut
ics.
Behd
ani
et a
l. 20
12
Lept
in r
ecep
tor
Subd
omai
nN
euro
pept
ide
YB
lood
-bra
in b
arri
er
The
adip
ocyt
e-de
rived
cyt
okin
e le
ptin
act
s as a
met
abol
ic sw
itch,
con
nect
ing
the
body
’s m
etab
olism
to h
igh-
ener
gy c
onsu
min
g pr
oces
ses
such
as r
epro
duct
ion
and
imm
une
resp
onse
s. A
ccum
ulat
ing
evid
ence
sugg
ests
that
lept
in p
lays
a ro
le in
hum
an p
atho
logi
es, s
uch
as
auto
imm
une
dise
ases
and
can
cer,
thus
pro
vidi
ng a
ratio
nale
for t
he d
evel
opm
ent o
f lep
tin a
ntag
onist
s. In
the
pres
ent s
tudy
, we
gene
rate
d an
d ev
alua
ted
a pa
nel o
f neu
tral
izin
g na
nobo
dies
targ
etin
g th
e LR
(lep
tin re
cept
or).
A n
anob
ody
com
prise
s the
var
iabl
e do
mai
n of
the
natu
rally
occ
urrin
g sin
gle-
chai
n an
tibod
ies f
ound
in m
embe
rs o
f the
Cam
elid
ae fa
mily
. We
iden
tified
thre
e cl
asse
s of n
eutr
aliz
ing
nano
-bo
dies
targ
etin
g di
ffere
nt L
R su
bdom
ains
: i.e
. the
CRH
2 (c
ytok
ine
rece
ptor
hom
olog
y 2)
, Ig-
like
and
FNII
I (fib
rone
ctin
type
III)
dom
ains
. O
nly
nano
bodi
es d
irect
ed a
gain
st th
e C
RH2
dom
ain
inhi
bite
d le
ptin
bin
ding
. We
coul
d sh
ow th
at a
nan
obod
y th
at ta
rget
s the
Ig-li
ke
dom
ain
pote
ntly
inte
rfer
ed w
ith le
ptin
-dep
ende
nt re
gula
tion
of h
ypot
hala
mic
NPY
(neu
rope
ptid
e Y)
exp
ress
ion.
As a
con
sequ
ence
, dai
ly
intr
aper
itone
al in
ject
ion
incr
ease
d bo
dy w
eigh
t, bo
dy fa
t con
tent
, foo
d in
take
, liv
er si
ze a
nd se
rum
insu
lin le
vels
. All
of th
ese
char
acte
ris-
tics r
esem
ble
the
phen
otyp
e of
lept
in a
nd L
R-de
ficie
nt a
nim
als.
The
resu
lts o
f the
pre
sent
stud
y su
ppor
t pro
pose
d m
odel
s of t
he a
ctiv
ated
LR
com
plex
, and
dem
onst
rate
that
it is
pos
sible
to b
lock
LR
signa
lling
with
out a
ffect
ing
ligan
d bi
ndin
g. Th
ese
nano
bodi
es fo
rm n
ew to
ols
to st
udy
the
mec
hani
sms o
f BBB
(blo
od-b
rain
bar
rier)
lept
in tr
ansp
ort a
nd th
e eff
ect o
f LR
inhi
bitio
n in
dise
ase
mod
els.
Zab
eau
et a
l. 20
12
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
477
Car
cino
embr
yoni
c an
tige
n re
late
d ce
ll ad
hesi
on m
olec
ule
Can
cer
Car
cino
embr
yoni
c an
tigen
rela
ted
cell
adhe
sion
mol
ecul
e (C
EAC
AM
) 6 is
ove
r-ex
pres
sed
in d
iffer
ent t
ypes
of c
ance
r cel
ls. I
n ad
di-
tion,
it h
as a
lso
been
impl
icat
ed in
som
e in
fect
ious
dis
ease
s. Ta
rget
ing
this
mol
ecul
e by
an
antib
ody
mig
ht h
ave
appl
icat
ions
in d
iver
se
tum
or m
odel
s. Si
ngle
dom
ain
antib
ody
(sdA
b) is
bec
omin
g ve
ry u
sefu
l for
mat
in a
ntib
ody
engi
neer
ing
as p
oten
tial t
ools
for t
reat
-in
g ac
ute
and
chro
nic
dise
ase
cond
ition
s suc
h as
can
cer f
or b
oth
diag
nost
ic a
s wel
l as t
hera
peut
ic a
pplic
atio
n. G
ener
ally
, sdA
bs w
ith
good
affi
nity
are
isol
ated
from
an
imm
une
libra
ry. D
isco
very
of a
new
targ
et a
ntig
en w
ould
requ
ire a
new
imm
uniz
atio
n w
ith p
urifi
ed
antig
en w
hich
is n
ot a
lway
s eas
y. In
this
stud
y, w
e ha
ve is
olat
ed, b
y ph
age
disp
lay,
an sd
Ab
agai
nst C
EAC
AM
6 w
ith a
n affi
nity
of 5
nM
fr
om a
llam
a im
mun
ized
with
can
cer c
ells
. The
antib
ody
has g
ood
biop
hysi
cal p
rope
rtie
s, an
d it
bind
s to
the
cells
exp
ress
ing
the
targ
et
antig
en. F
urth
erm
ore,
it re
duce
s can
cer c
ells
prol
ifera
tion
in v
itro
and
show
s an
exce
llent
tum
or ta
rget
ing
in v
ivo.
This
sdA
b co
uld
be
usef
ul in
dia
gnos
is a
s wel
l as t
hera
py o
f CEA
CA
M6
expr
essi
ng tu
mor
s. Fi
nally
, we
envi
sage
it w
ould
be
feas
ible
to is
olat
e go
od sd
Abs
ag
ains
t oth
er in
tere
stin
g tu
mor
ass
ocia
ted
antig
ens f
rom
this
libr
ary.
Ther
efor
e, th
is im
mun
izat
ion
met
hod
coul
d be
a g
ener
al st
rate
gy
for i
sola
ting
sdA
bs a
gain
st a
ny su
rfac
e an
tigen
with
out i
mm
uniz
ing
the
anim
al w
ith th
e an
tigen
of i
nter
est e
ach
time.
Bara
l et
al. 2
011
12 n
anob
ody
clon
esO
ligoc
lona
l nan
obod
ies
Bre
ast c
ance
r ce
llsH
ER2
Mod
ern
anti-
HER
2 an
tibod
y th
erap
y te
nds t
o ex
ploi
t a p
anel
of d
iffer
ent a
ntib
odie
s aga
inst
diff
eren
t epi
tope
s on
the
antig
en. F
or
this
aim
, nan
obod
ies a
re v
ery
stri
king
targ
etin
g ag
ents
and
can
be
easi
ly p
rodu
ced
agai
nst a
ny c
ell-s
peci
fic m
embr
ane
antig
en. Th
e ol
igoc
lona
l nan
obod
ies c
an b
e us
ed to
blo
ck m
ore
than
one
func
tiona
l epi
tope
on
a ta
rget
ant
igen
and
inhi
bit t
he g
ener
atio
n of
esc
ape
vari
ants
ass
ocia
ted
with
can
cer t
hera
py. I
n th
is st
udy,
12 n
anob
ody
clon
es se
lect
ed fr
om a
n im
mun
e ca
mel
libr
ary
wer
e ex
amin
ed fo
r th
eir a
bilit
y to
diff
er b
etw
een
tum
or m
arke
rs. Th
ese
olig
oclo
nal n
anob
odie
s tar
gete
d br
east
can
cer c
ells
bette
r tha
n ea
ch in
divi
dual
na
nobo
dy. I
n ep
itope
map
ping
, sev
eral
nan
obod
ies o
verla
pped
in th
e ep
itope
reco
gniz
ed b
y tr
astu
zum
ab a
nd so
me
of th
e no
n-ov
er-
lapp
ing
nano
bodi
es c
ould
affe
ct th
e bi
ndin
g of
tras
tuzu
mab
to H
ER2.
This
stud
y de
mon
stra
tes t
hat t
he o
ligoc
lona
l nan
obod
ies a
re
pote
ntia
l the
rape
utic
tool
s tha
t can
be
used
inst
ead
of, o
r in
com
bina
tion
with
tras
tuzu
mab
to a
sses
s tum
or v
iabi
lity
duri
ng tr
eatm
ent.
Jam
nani
et
al.
2012
Ion
chan
nel M
2In
fluen
za A
Influ
enza
A v
irus
pos
es se
riou
s hea
lth th
reat
to h
uman
s. N
eutr
aliz
ing
antib
odie
s aga
inst
the
high
ly c
onse
rved
M2
ion
chan
nel i
s th
ough
t to
offer
bro
ad p
rote
ctio
n ag
ains
t infl
uenz
a A
vir
uses
. Her
e, w
e sc
reen
ed sy
nthe
tic C
amel
sing
le-d
omai
n an
tibod
y (V
HH
) lib
rari
es a
gain
st n
ativ
e M
2 io
n ch
anne
l pro
tein
. One
of t
he is
olat
ed V
HH
s, M
2-7A
, spe
cific
ally
bou
nd to
M2-
expr
esse
d ce
ll m
em-
bran
e as
wel
l as i
nflue
nza
A v
irio
n, in
hibi
ted
repl
icat
ion
of b
oth
aman
tadi
ne-s
ensi
tive
and
resi
stan
t infl
uenz
a A
vir
uses
in v
itro,
and
pr
otec
ted
mic
e fr
om a
leth
al in
fluen
za v
irus
cha
lleng
e. M
oreo
ver,
M2-
7A sh
owed
blo
ckin
g ac
tivity
for p
roto
n in
flux
thro
ugh
M2
ion
chan
nel.
Thes
e pi
eces
of e
vide
nce
colle
ctiv
ely
dem
onst
rate
for t
he fi
rst t
ime
that
a n
eutr
aliz
ing
antib
ody
agai
nst M
2 w
ith b
road
sp
ecifi
city
is a
chie
vabl
e, a
nd M
2-7A
may
hav
e po
tent
ial f
or c
ross
pro
tect
ion
agai
nst a
num
ber o
f var
iant
s and
subt
ypes
of i
nflue
nza
A
viru
ses.
Wei
et a
l. 20
11
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
478
Tabl
e 6C
. App
licat
ion
of s
ingl
e-do
mai
n an
tibo
dy fr
agm
ents
in th
erap
y: p
atho
gen
neut
rali
sati
on
Biv
alen
t nan
obod
yIn
fluen
za v
irus
In
tran
asal
ad
min
istr
atio
n
Influ
enza
A v
irus
infe
ctio
ns im
pose
a re
curr
ent a
nd g
loba
l dis
ease
bur
den.
Cur
rent
ant
ivir
als a
gain
st in
fluen
za a
re n
ot a
lway
s eff
ectiv
e. W
e as
sess
ed th
e pr
otec
tive
pote
ntia
l of m
onov
alen
t and
biv
alen
t Nan
obod
ies (
Abl
ynx)
aga
inst
cha
lleng
e w
ith th
is v
irus
. Th
ese
Nan
obod
ies w
ere
deri
ved
from
llam
as a
nd ta
rget
H5N
1 he
mag
glut
inin
. Int
rana
sal a
dmin
istr
atio
n of
Nan
obod
ies e
ffect
ivel
y co
ntro
lled
hom
olog
ous i
nflue
nza
A v
irus
repl
icat
ion.
Adm
inis
trat
ion
of N
anob
odie
s bef
ore
chal
leng
e st
rong
ly re
duce
d H
5N1
viru
s re
plic
atio
n in
the
lung
s and
pro
tect
ed m
ice
from
mor
bidi
ty a
nd m
orta
lity
afte
r a le
thal
cha
lleng
e w
ith H
5N1
viru
s. Th
e bi
vale
nt
Nan
obod
y w
as a
t lea
st 6
0-fo
ld m
ore
effec
tive
than
the
mon
oval
ent N
anob
ody
in c
ontr
ollin
g vi
rus r
eplic
atio
n. In
add
ition
, Nan
o-bo
dy th
erap
y af
ter c
halle
nge
stro
ngly
redu
ced
vira
l rep
licat
ion
and
sign
ifica
ntly
del
ayed
tim
e to
dea
th. E
pito
pe m
appi
ng re
veal
ed
that
the
VH
H N
anob
ody
bind
s to
antig
enic
site
B in
H5
hem
aggl
utin
in. B
ecau
se N
anob
odie
s are
smal
l, st
able
, and
sim
ple
to p
ro-
duce
, the
y ar
e a
prom
isin
g, n
ovel
ther
apeu
tic a
gent
aga
inst
influ
enza
.
Iban
ez e
t al
. 201
1
Mul
tim
eric
/bis
peci
fic
cons
truc
tsIn
fluen
za
For e
ffici
ent p
reve
ntio
n of
vir
al in
fect
ions
and
cro
ss p
rote
ctio
n, si
mul
tane
ous t
arge
ting
of m
ultip
le v
iral
epi
tope
s is a
pow
er-
ful s
trat
egy.
Llam
a he
avy
chai
n an
tibod
y fr
agm
ents
(VH
H) a
gain
st th
e tr
imer
ic e
nvel
ope
prot
eins
of R
espi
rato
ry S
yncy
tial V
irus
(F
usio
n pr
otei
n), R
abie
s vir
us (G
lyco
prot
ein)
and
H5N
1 In
fluen
za (H
emag
glut
inin
5) w
ere
sele
cted
from
llam
a de
rive
d im
mun
e lib
rari
es b
y ph
age
disp
lay.
Neu
tral
izin
g V
HH
reco
gniz
ing
diffe
rent
epi
tope
s in
the
rece
ptor
bin
ding
site
s on
the
spik
es w
ith a
ffini
-tie
s in
the
low
nan
omol
ar ra
nge
wer
e id
entifi
ed fo
r all
the
thre
e vi
ruse
s by
vira
l neu
tral
izat
ion
assa
ys. B
y fu
sion
of V
HH
with
var
i-ab
le li
nker
leng
ths,
mul
timer
ic c
onst
ruct
s wer
e m
ade
that
impr
oved
neu
tral
izat
ion
pote
ncie
s up
to 4
000-
fold
for R
SV, 1
500-
fold
fo
r Rab
ies v
irus
and
75-
fold
for I
nflue
nza
H5N
1. Th
e po
tenc
ies o
f the
VH
H c
onst
ruct
s wer
e si
mila
r or b
ette
r tha
n be
st p
erfo
rmin
g m
onoc
lona
l ant
ibod
ies.
The
cros
s pro
tect
ion
capa
city
aga
inst
diff
eren
t vir
al st
rain
s was
als
o im
prov
ed fo
r all
thre
e vi
ruse
s, b
oth
by
mul
tival
ent (
two
or th
ree
iden
tical
VH
H) a
nd b
ipar
atop
ic (t
wo
diffe
rent
VH
H) c
onst
ruct
s. B
y co
mbi
ning
a V
HH
neu
tral
izin
g RS
V
subt
ype
A, b
ut n
ot su
btyp
e B
with
a p
oorl
y ne
utra
lizin
g V
HH
with
hig
h affi
nity
for s
ubty
pe B
, a b
ipar
atop
ic c
onst
ruct
was
mad
e w
ith lo
w n
anom
olar
neu
tral
izin
g po
tenc
y ag
ains
t bot
h su
btyp
es. T
riva
lent
ant
i-H5N
1 V
HH
neu
tral
ized
bot
h In
fluen
za H
5N1
clad
e1 a
nd 2
in a
pse
udot
ype
assa
y an
d w
as v
ery
pote
nt in
neu
tral
izin
g th
e N
IBRG
-14
Influ
enza
H5N
1 st
rain
with
IC(5
0) o
f nin
e pi
com
olar
. Biv
alen
t and
bip
arat
opic
con
stru
cts a
gain
st R
abie
s vir
us c
ross
neu
tral
ized
bot
h 10
diff
eren
t Gen
otyp
e 1
stra
ins a
nd
Gen
otyp
e 5.
The
resu
lts sh
ow th
at m
ultim
eriz
atio
n of
VH
H fr
agm
ents
targ
etin
g m
ultip
le e
pito
pes o
n a
vira
l tri
mer
ic sp
ike
prot
ein
is a
pow
erfu
l too
l for
ant
i-vi
ral t
hera
py to
ach
ieve
“bes
t-in
-cla
ss” a
nd b
road
er n
eutr
aliz
atio
n ca
paci
ty.
Hul
tber
g et
al.
2011
HIV
vir
usC
XC
R4
Epit
ope
map
ping
The
impo
rtan
t fam
ily o
f G p
rote
in-c
oupl
ed re
cept
ors h
as so
far n
ot b
een
targ
eted
ver
y su
cces
sful
ly w
ith c
onve
ntio
nal m
onoc
lona
l an
tibod
ies.
Her
e w
e re
port
the
isol
atio
n an
d ch
arac
teri
zatio
n of
func
tiona
l VH
H-b
ased
imm
unog
lobu
lin si
ngle
var
iabl
e do
mai
ns
(or n
anob
odie
s) a
gain
st th
e ch
emok
ine
rece
ptor
CX
CR4
. Tw
o hi
ghly
sele
ctiv
e m
onov
alen
t nan
obod
ies,
238
D2
and
238D
4, w
ere
obta
ined
usi
ng a
tim
e-effi
cien
t who
le c
ell i
mm
uniz
atio
n, p
hage
dis
play
, and
cou
nter
sele
ctio
n m
etho
d. Th
e hi
ghly
sele
ctiv
e V
HH
-ba
sed
imm
unog
lobu
lin si
ngle
var
iabl
e do
mai
ns c
ompe
titiv
ely
inhi
bite
d th
e C
XC
R4-m
edia
ted
sign
alin
g an
d an
tago
nize
d th
e ch
emoa
ttra
ctan
t effe
ct o
f the
CX
CR4
liga
nd C
XC
L12.
Epi
tope
map
ping
show
ed th
at th
e tw
o na
nobo
dies
bin
d to
dis
tinct
but
pa
rtia
lly o
verl
appi
ng si
tes i
n th
e ex
trac
ellu
lar l
oops
. Sho
rt p
eptid
e lin
kage
of 2
38D
2 w
ith 2
38D
4 re
sulte
d in
sign
ifica
ntly
incr
ease
d affi
nity
for C
XC
R4 a
nd p
icom
olar
act
ivity
in a
ntic
hem
otac
tic a
ssay
s. In
tere
stin
gly,
the
mon
oval
ent n
anob
odie
s beh
aved
as n
eutr
al
anta
goni
sts,
whe
reas
the
bipa
rato
pic
nano
bodi
es a
cted
as i
nver
se a
goni
sts a
t the
con
stitu
tivel
y ac
tive
CX
CR4
-N3.
35A
. The
CX
CR4
na
nobo
dies
dis
play
ed st
rong
ant
iret
rovi
ral a
ctiv
ity a
gain
st T
cel
l-tr
opic
and
dua
l-tr
opic
HIV
-1 st
rain
s. M
oreo
ver,
the
bipa
rato
pic
nano
body
effe
ctiv
ely
mob
ilize
d C
D34
-pos
itive
stem
cel
ls in
cyn
omol
gus m
onke
ys. Th
us, t
he n
anob
ody
plat
form
may
be
high
ly
effec
tive
at g
ener
atin
g ex
trem
ely
pote
nt a
nd se
lect
ive
G p
rote
in-c
oupl
ed re
cept
or m
odul
ator
s.
Jah-
nich
en e
t al
. 201
0
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
479
HIV
vir
usEn
velo
pe p
rote
inC
D4
rece
ptor
Man
y of
the
neut
ralis
ing
antib
odie
s, is
olat
ed to
dat
e, d
ispl
ay li
mite
d ac
tiviti
es a
gain
st th
e gl
obal
ly m
ost p
reva
lent
HIV
-1 su
btyp
es
A a
nd C
. Ther
efor
e, th
ose
subt
ypes
are
con
side
red
to b
e an
impo
rtan
t tar
get f
or a
ntib
ody-
base
d th
erap
y. Va
riab
le d
omai
ns o
f lla
ma
heav
y ch
ain
antib
odie
s (V
HH
) hav
e so
me
supe
rior
pro
pert
ies c
ompa
red
with
cla
ssic
al a
ntib
odie
s. Th
eref
ore
we
desc
ribe
the
appl
icat
ion
of tr
imer
ic fo
rms o
f env
elop
e pr
otei
ns (E
nv),
deri
ved
from
HIV
-1 o
f sub
type
A a
nd B
/C, f
or a
pro
long
ed im
mun
izat
ion
of tw
o lla
mas
. A p
anel
of V
HH
, whi
ch in
terf
ere
with
CD
4 bi
ndin
g to
HIV
-1 E
nv w
ere
sele
cted
with
use
of p
anni
ng. Th
e re
sults
of
bind
ing
and
com
petit
ion
assa
ys to
var
ious
Env
, inc
ludi
ng a
var
iant
with
a st
abili
zed
CD
4-bi
ndin
g st
ate
(gp1
20(D
s2))
, cro
ss-c
ompe
-tit
ion
expe
rim
ents
, mat
urat
ion
anal
ysis
and
neu
tral
isat
ion
assa
ys, e
nabl
ed u
s to
clas
sify
the
sele
cted
VH
H in
to th
ree
grou
ps. Th
e V
HH
of g
roup
I w
ere
effici
ent m
ainl
y ag
ains
t vir
uses
of s
ubty
pe A
, C a
nd B
’/C. Th
e V
HH
of g
roup
II re
sem
ble
the
broa
dly
neu-
tral
isin
g an
tibod
y (b
nmA
b) b
12, n
eutr
aliz
ing
mai
nly
subt
ype
B an
d C
vir
uses
, how
ever
som
e ha
d a
broa
der n
eutr
alis
atio
n pr
ofile
. A
repr
esen
tativ
e of
the
thir
d gr
oup,
2E7
, had
an
even
hig
her n
eutr
aliz
atio
n br
eadt
h, n
eutr
aliz
ing
21 o
ut o
f the
26
test
ed st
rain
s be
long
ing
to th
e A
, A/G
, B, B
/C a
nd C
subt
ypes
. To
eval
uate
the
cont
ribu
tion
of c
erta
in a
min
o ac
ids t
o th
e po
tenc
y of
the
VH
H
a sm
all s
et o
f the
mut
ants
wer
e co
nstr
ucte
d. S
urpr
isin
gly
this
yie
lded
one
mut
ant w
ith sl
ight
ly im
prov
ed n
eutr
alis
atio
n po
tenc
y ag
ains
t 92U
G37
.A9
(sub
type
A) a
nd 9
6ZM
651.
02 (s
ubty
pe C
). Th
ese
findi
ngs a
nd th
e w
ell-k
now
n st
abili
ty o
f VH
H in
dica
te th
e po
tent
ial a
pplic
atio
n of
thes
e V
HH
as a
nti-H
IV-1
mic
robi
cide
s.
Stro
kap-
pe
et a
l. 20
12
Rot
avir
usG
astr
oent
erit
isIm
mun
ocap
ture
ele
ctro
n m
icro
scop
y
Rota
viru
s is t
he m
ain
caus
e of
vir
al g
astr
oent
eriti
s in
youn
g ch
ildre
n. Th
eref
ore,
the
deve
lopm
ent o
f ine
xpen
sive
ant
ivir
al p
rod-
ucts
for t
he p
reve
ntio
n an
d/or
trea
tmen
t of r
otav
irus
dis
ease
rem
ains
a p
rior
ity. P
revi
ousl
y w
e ha
ve sh
own
that
a re
com
bina
nt
mon
oval
ent a
ntib
ody
frag
men
t (re
ferr
ed to
as A
nti-R
otav
irus
Pro
tein
s or A
RP1)
der
ived
from
a h
eavy
cha
in a
ntib
ody
of a
llam
a im
mun
ised
with
rota
viru
s was
abl
e to
neu
tral
ise
rota
viru
s inf
ectio
n in
a m
ouse
mod
el sy
stem
. In
the
pres
ent w
ork
we
inve
stig
ated
th
e sp
ecifi
city
and
neu
tral
isin
g ac
tivity
of t
wo
llam
a an
tibod
y fr
agm
ents
, ARP
1 an
d A
RP3,
aga
inst
13
cell
cultu
re a
dapt
ed ro
tavi
-ru
s str
ains
of d
iver
se g
enot
ypes
. In
addi
tion,
imm
unoc
aptu
re e
lect
ron
mic
rosc
opy
(IEM
) was
per
form
ed to
det
erm
ine
bind
ing
of
ARP
1 to
clin
ical
isol
ates
and
cel
l cul
ture
ada
pted
stra
ins.
ARP
1 an
d A
RP3
wer
e ab
le to
neu
tral
ise
a br
oad
vari
ety
of ro
tavi
rus s
ero-
type
s/ge
noty
pes i
n vi
tro,
and
in a
dditi
on, I
EM sh
owed
spec
ific
bind
ing
to a
var
iety
of c
ell a
dapt
ed st
rain
s as w
ell a
s str
ains
from
cl
inic
al sp
ecim
ens.
Thes
e re
sults
indi
cate
d th
at th
ese
mol
ecul
es c
ould
pot
entia
lly b
e us
ed a
s im
mun
opro
phyl
actic
and
/or i
mm
uno-
ther
apeu
tic p
rodu
cts f
or th
e pr
even
tion
and/
or t
reat
men
t of i
nfec
tion
of a
bro
ad ra
nge
of c
linic
ally
rele
vant
rota
viru
s str
ains
.
Ala
din
et
al. 2
012
Plas
mod
ium
viv
axFy
blo
od g
roup
Duff
y an
tige
n re
cept
or
for
chem
okin
es
Fy b
lood
gro
up a
ntig
ens a
re c
arri
ed b
y th
e D
uffy
antig
en re
cept
or fo
r che
mok
ines
(DA
RC),
a re
d ce
lls re
cept
or fo
r Pla
smod
ium
vi
vax
broa
dly
impl
icat
ed in
hum
an h
ealth
and
dis
ease
s. R
ecom
bina
nt V
HH
s, o
r nan
obod
ies,
the
smal
lest
inta
ct a
ntig
en b
ind-
ing
frag
men
t der
ivat
ive
from
the
heav
y ch
ain-
only
ant
ibod
ies p
rese
nt in
cam
elid
s, w
ere
prep
ared
from
a d
rom
edar
y im
mun
ized
ag
ains
t DA
RC N
-ter
min
al e
xtra
cellu
lar d
omai
n an
d se
lect
ed fo
r DA
RC b
indi
ng. A
des
crib
ed V
HH
, CA
52, d
oes r
ecog
nize
nat
ive
DA
RC o
n ce
lls. I
t inh
ibits
P. v
ivax
inva
sion
of e
ryth
rocy
tes a
nd d
ispl
aces
inte
rleu
kin-
8 bo
und
to D
ARC
. The
targ
eted
epi
tope
ov
erla
ps th
e w
ell-d
efine
d D
ARC
Fy6
epi
tope
. K(D
) of C
A52
-DA
RC e
quili
briu
m is
sub-
nano
mol
ar, h
ence
idea
l to
deve
lop
diag
nos-
tic o
r the
rape
utic
com
poun
ds. I
mm
unoc
aptu
re b
y im
mob
ilize
d C
A52
yie
lded
hig
hly
puri
fied
DA
RC fr
om e
ngin
eere
d K
562
cells
. Th
is fi
rst r
epor
t on
a V
HH
with
spec
ifici
ty fo
r a re
d bl
ood
cell
prot
ein
exem
plifi
es V
HH
s’ po
tent
ialit
ies t
o ta
rget
, to
puri
fy, a
nd to
m
odul
ate
the
func
tion
of c
ellu
lar m
arke
rs.
Smol
arek
et
al.
2010
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
480
Tryp
anos
oma
bruc
eiVa
rian
t-sp
ecifi
c su
rfac
e gl
ycop
rote
inN
anob
ody-
med
iate
d ly
sis
The
Afr
ican
tryp
anos
ome
Tryp
anos
oma
bruc
ei, w
hich
per
sist
s with
in th
e bl
oods
trea
m o
f the
mam
mal
ian
host
, has
evo
lved
pot
ent
mec
hani
sms f
or im
mun
e ev
asio
n. S
peci
fical
ly, a
ntig
enic
var
iatio
n of
the
vari
ant-
spec
ific
surf
ace
glyc
opro
tein
(VSG
) and
a h
ighl
y ac
tive
endo
cyto
sis a
nd re
cycl
ing
of th
e su
rfac
e co
at e
ffici
ently
del
ay k
illin
g m
edia
ted
by a
nti-V
SG a
ntib
odie
s. C
onse
quen
tly, c
on-
vent
iona
l VSG
-spe
cific
inta
ct im
mun
oglo
bulin
s are
non
-try
pano
cida
l in
the
abse
nce
of c
ompl
emen
t. In
shar
p co
ntra
st, m
onov
a-le
nt a
ntig
en-b
indi
ng fr
agm
ents
, inc
ludi
ng 1
5 kD
a na
nobo
dies
(Nb)
der
ived
from
cam
elid
hea
vy-c
hain
ant
ibod
ies (
HC
Abs
) rec
og-
nizi
ng v
aria
nt-s
peci
fic V
SG e
pito
pes,
effi
cien
tly ly
se tr
ypan
osom
es b
oth
in v
itro
and
in v
ivo.
This
Nb-
med
iate
d ly
sis i
s pre
cede
d by
ver
y ra
pid
imm
obili
satio
n of
the
para
site
s, m
assi
ve e
nlar
gem
ent o
f the
flag
ella
r poc
ket a
nd m
ajor
blo
ckad
e of
end
ocyt
osis
. This
is
acc
ompa
nied
by
seve
re m
etab
olic
per
turb
atio
ns re
flect
ed b
y re
duce
d in
trac
ellu
lar A
TP-
leve
ls a
nd lo
ss o
f mito
chon
dria
l mem
-br
ane
pote
ntia
l, cu
lmin
atin
g in
cel
l dea
th. M
odifi
catio
n of
ant
i-VSG
Nbs
thro
ugh
site
-dir
ecte
d m
utag
enes
is a
nd b
y re
cons
titut
ion
into
HC
Abs
, com
bine
d w
ith u
nvei
ling
of tr
ypan
olyt
ic a
ctiv
ity fr
om in
tact
imm
unog
lobu
lins b
y pa
pain
pro
teol
ysis
, dem
onst
rate
s th
at th
e tr
ypan
olyt
ic a
ctiv
ity o
f Nbs
and
Fab
s req
uire
s low
mol
ecul
ar w
eigh
t, m
onov
alen
cy a
nd h
igh
affini
ty. W
e pr
opos
e th
at th
e ge
nera
tion
of lo
w m
olec
ular
wei
ght V
SG-s
peci
fic tr
ypan
olyt
ic n
anob
odie
s tha
t im
pede
end
ocyt
osis
offe
rs a
new
opp
ortu
nity
for
deve
lopi
ng n
ovel
tryp
anos
omia
sis t
hera
peut
ics.
In a
dditi
on, t
hese
dat
a su
gges
t tha
t the
ant
igen
-bin
ding
dom
ain
of a
n an
ti-m
icro
-bi
al a
ntib
ody
harb
ours
bio
logi
cal f
unct
iona
lity
that
is la
tent
in th
e in
tact
imm
unog
lobu
lin a
nd is
reve
aled
onl
y up
on re
leas
e of
the
antig
en-b
indi
ng fr
agm
ent.
Stijl
e-m
ans e
t al
. 201
1
Sial
idas
eTr
ypan
osom
a cr
uzi
The
sial
ic a
cid
pres
ent i
n th
e pr
otec
tive
surf
ace
muc
in c
oat o
f Try
pano
som
a cr
uzi i
s add
ed b
y a
mem
bran
e an
chor
ed tr
ans-
sial
idas
e (T
cTS)
, a m
odifi
ed si
alid
ase
that
is e
xpre
ssed
from
a la
rge
gene
fam
ily. I
n th
is w
ork,
we
anal
yzed
sing
le d
omai
n ca
mel
id
antib
odie
s pro
duce
d ag
ains
t tra
ns-s
ialid
ase.
Lla
mas
wer
e im
mun
ized
with
a re
com
bina
nt tr
ans-
sial
idas
e an
d in
hibi
tory
sing
le-
dom
ain
antib
ody
frag
men
ts w
ere
obta
ined
by
phag
e di
spla
y se
lect
ion,
taki
ng a
dvan
tage
of a
scre
enin
g st
rate
gy u
sing
an
inhi
bitio
n te
st in
stea
d of
the
clas
sic
bind
ing
assa
y. Fo
ur si
ngle
dom
ain
antib
odie
s dis
play
ing
stro
ng tr
ans-
sial
idas
e in
hibi
tion
activ
ity a
gain
st
the
reco
mbi
nant
enz
yme
wer
e id
entifi
ed. Th
ey sh
are
the
sam
e co
mpl
emen
tari
ty-d
eter
min
ing
regi
on 3
leng
th (1
7 re
sidu
es) a
nd
have
ver
y si
mila
r seq
uenc
es. Th
is re
sult
indi
cate
s tha
t the
y lik
ely
deri
ved
from
a u
niqu
e cl
one.
Pro
babl
y th
ere
is o
nly
one
stru
ctur
al
solu
tion
for t
ight
bin
ding
inhi
bito
ry a
ntib
odie
s aga
inst
the
TcT
S us
ed fo
r im
mun
izat
ion.
To
our s
urpr
ise,
this
sing
le d
omai
n an
ti-bo
dy th
at in
hibi
ts th
e re
com
bina
nt T
cTS,
faile
d to
inhi
bit t
he e
nzym
atic
act
ivity
pre
sent
in p
aras
ite e
xtra
cts.
Ana
lysi
s of i
ndiv
idua
l re
com
bina
nt tr
ans-
sial
idas
es sh
owed
that
enz
ymes
exp
ress
ed fr
om d
iffer
ent g
enes
wer
e in
hibi
ted
to d
iffer
ent e
xten
ts (f
rom
8 to
98
%) b
y th
e lla
ma
antib
odie
s. A
min
o ac
id c
hang
es a
t key
pos
ition
s are
like
ly to
be
resp
onsi
ble
for t
he d
iffer
ence
s in
inhi
bitio
n fo
und
amon
g th
e re
com
bina
nt e
nzym
es. Th
ese
resu
lts su
gges
t tha
t the
pre
senc
e of
a la
rge
and
dive
rse
tran
s-si
alid
ase
fam
ily m
ight
be
requ
ired
to p
reve
nt th
e in
hibi
tory
resp
onse
aga
inst
this
ess
entia
l enz
yme
and
mig
ht th
us c
onst
itute
a n
ovel
stra
tegy
of T
. cru
zi
to e
vade
the
host
imm
une
syst
em.
Ratie
r et
al. 2
008
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
481
Ant
igen
var
iati
onVa
rian
t sur
face
gl
ycop
rote
ins
Cry
ptic
/con
serv
ed
epit
opes
Tryp
anos
omos
is th
erap
yA
ntib
ody:
bet
a-la
ctam
ase
fusi
on
Ant
igen
var
iatio
n is
a su
cces
sful
def
ense
syst
em a
dopt
ed b
y se
vera
l inf
ectio
us a
gent
s to
evad
e th
e ho
st im
mun
e re
spon
se. Th
e pr
inci
ple
of th
is d
efen
se st
rate
gy in
the
Afr
ican
tryp
anos
ome
para
digm
invo
lves
a d
ense
pac
king
of v
aria
nt su
rfac
e gl
ycop
rote
ins
(VSG
) exp
osin
g on
ly h
ighl
y va
riab
le a
nd im
mun
o-do
min
ant e
pito
pes t
o th
e im
mun
e sy
stem
, whe
reas
con
serv
ed e
pito
pes b
ecom
e in
acce
ssib
le fo
r lar
ge m
olec
ules
. Red
ucin
g th
e si
ze o
f bin
ders
that
targ
et th
e co
nser
ved,
less
-im
mun
ogen
ic, c
rypt
ic V
SG e
pito
pes
form
s an
obvi
ous s
olut
ion
to c
omba
t the
se p
aras
ites.
This
goa
l was
ach
ieve
d by
intr
oduc
ing
drom
edar
y H
eavy
-cha
in a
ntib
odie
s. W
e fo
und
that
onl
y th
ese
uniq
ue a
ntib
odie
s rec
ogni
ze e
pito
pes c
omm
on to
mul
tiple
VSG
cla
sses
. Aft
er p
hage
dis
play
of t
heir
an
tigen
-bin
ding
repe
rtoi
re, w
e is
olat
ed a
sing
le d
omai
n an
tibod
y fr
agm
ent w
ith h
igh
spec
ifici
ty fo
r the
con
serv
ed A
sn-li
nked
ca
rboh
ydra
te o
f VSG
. In
shar
p co
ntra
st to
labe
led
conc
anav
alin
-A th
at st
ains
onl
y th
e fla
gella
r poc
ket w
here
car
bohy
drat
es a
re
acce
ssib
le b
ecau
se o
f les
s den
se V
SG p
acki
ng, t
he si
ngle
dom
ain
bind
er st
ains
the
entir
e su
rfac
e of
via
ble
para
site
s, ir
resp
ectiv
e of
th
e V
SG ty
pe e
xpre
ssed
. This
cor
robo
rate
s the
idea
that
smal
l ant
ibod
y fr
agm
ents
, but
not
larg
er le
ctin
s or c
onve
ntio
nal a
ntib
ody
frag
men
ts, a
re a
ble
to p
enet
rate
the
dens
e V
SG c
oat t
o ta
rget
thei
r epi
tope
. The
diag
nost
ic p
oten
tial o
f thi
s fluo
resc
ently
labe
led
bind
er w
as p
rove
n by
the
dire
ct, s
elec
tive,
and
sens
itive
det
ectio
n of
par
asite
s in
bloo
d sm
ears
. The
empl
oym
ent o
f thi
s bin
der a
s a
mol
ecul
ar re
cogn
ition
uni
t in
imm
unot
oxin
s des
igne
d fo
r try
pano
som
osis
ther
apy
beco
mes
feas
ible
as w
ell.
This
was
illu
stra
ted
by
the
spec
ific
tryp
anol
ysis
indu
ced
by a
n an
tibod
y: b
eta-
lact
amas
e fu
sion
act
ivat
ing
a pr
odru
g.
Stijl
e-m
ans e
t al
. 200
4
Cry
ptic
epi
tope
sA
pica
l mem
bran
e
anti
gen
1Pl
asm
odiu
m fa
lcip
arur
mEx
tens
ive
poly
mor
phis
mIg
NA
R
Api
cal m
embr
ane
antig
en 1
(AM
A1)
is e
ssen
tial f
or in
vasi
on o
f ery
thro
cyte
s and
hep
atoc
ytes
by
Plas
mod
ium
par
asite
s and
is
a le
adin
g m
alar
ial v
acci
ne c
andi
date
. Alth
ough
con
vent
iona
l ant
ibod
ies t
o A
MA
1 ca
n pr
even
t suc
h in
vasi
on, e
xten
sive
pol
y-m
orph
ism
s with
in su
rfac
e-ex
pose
d lo
ops m
ay li
mit
the
abili
ty o
f the
se A
MA
1 in
duce
d an
tibod
ies t
o pr
otec
t aga
inst
all
para
site
ge
noty
pes.
Usi
ng a
n A
MA
1-sp
ecifi
c Ig
NA
R si
ngle
-var
iabl
e-do
mai
n an
tibod
y, w
e pe
rfor
med
targ
eted
mut
agen
esis
and
sele
ctio
n ag
ains
t AM
A1
from
thre
e P.
falc
ipar
urm
stra
ins.
We
pres
ent c
ocry
stal
stru
ctur
es o
f tw
o an
tibod
y-A
MA
1 co
mpl
exes
whi
ch re
veal
ex
tend
ed Ig
NA
R C
DR3
loop
s pen
etra
ting
deep
into
a h
ydro
phob
ic c
left
on
the
antig
en su
rfac
e an
d co
ntac
ting
resi
dues
con
serv
ed
acro
ss p
aras
ite sp
ecie
s. C
ompa
riso
n of
a se
ries
of a
ffini
ty-e
nhan
cing
mut
atio
ns a
llow
ed d
isse
ctio
n of
thei
r rel
ativ
e co
ntri
butio
ns
to b
indi
ng k
inet
ics a
nd c
orre
latio
n w
ith in
hibi
tion
of e
ryth
rocy
te in
vasi
on. Th
ese
findi
ngs p
rovi
de in
sigh
ts in
to m
echa
nism
s of
sing
le-d
omai
n an
tibod
y bi
ndin
g, a
nd m
ay e
nabl
e de
sign
of r
eage
nts t
arge
ting
othe
rwis
e cr
yptic
epi
tope
s in
path
ogen
ant
igen
s.
Hen
der-
son
et a
l. 20
07
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
482
Tabl
e 6D
. App
licat
ion
of s
ingl
e-do
mai
n an
tibo
dy fr
agm
ents
in th
erap
y: in
trac
ellu
lar
expr
essi
on o
f sin
gle-
dom
ain
anti
body
frag
men
ts
Apo
ptos
isC
aspa
se 3
Neu
rode
gene
rati
ve
dise
ases
Can
cer
Apo
ptos
is, o
r pro
gram
med
cel
l dea
th, i
s an
esse
ntia
l pro
cess
affe
ctin
g ho
meo
stas
is o
f cel
l gro
wth
, dev
elop
men
t, an
d th
e el
imin
a-tio
n of
dam
aged
or d
ange
rous
cel
ls. I
napp
ropr
iate
cel
l dea
th c
ause
d by
oxi
dativ
e st
ress
has
bee
n im
plic
ated
in th
e de
velo
pmen
t of
neu
rode
gene
rativ
e di
seas
es su
ch a
s Alz
heim
er’s,
Par
kins
on’s,
and
stro
ke. O
n th
e ot
her h
and,
a d
efec
t in
the
cell
deat
h pr
oces
s le
ads t
o th
e de
velo
pmen
t of c
ance
r. Fo
r exa
mpl
e, th
e m
ain
play
er o
f apo
ptos
is, p
53, i
s def
ectiv
e in
man
y of
the
hum
an c
ance
rs.
Apo
ptos
is is
regu
late
d by
the
inte
rpla
y of
pro
-apo
ptot
ic a
nd a
nti-a
popt
otic
pro
tein
s fro
m th
e Bc
l-2 fa
mily
and
cas
pase
s. In
par
-tic
ular
, spe
cific
mod
ulat
ors o
f the
act
ivity
of C
aspa
se 3
cou
ld b
e ve
ry im
port
ant f
or th
e de
velo
pmen
t of t
hera
pies
for d
isea
ses s
uch
as n
euro
dege
nera
tion
and
canc
er. I
n th
is st
udy,
two
V(H
)Hs s
peci
fic to
Cas
pase
3 (V
hhC
asp3
1 an
d V
hhC
asp3
2) w
ere
isol
ated
fr
om a
hea
vy c
hain
ant
ibod
y va
riab
le d
omai
n (V
(H)H
) pha
ge d
ispl
ay li
brar
y an
d te
sted
for t
heir
apo
ptos
is-m
odul
atin
g eff
ects
. W
hile
Vhh
Cas
p31
was
foun
d to
be
anta
goni
stic
tow
ards
Cas
pase
3, V
hhC
asp3
2 w
as a
goni
stic
. Fur
ther
mor
e, w
hen
expr
esse
d as
in
trab
odie
s in
SHSY
-5Y
neur
obla
stom
a ce
lls, V
hhC
asp3
1 re
nder
ed c
ells
resi
stan
t to
oxid
ativ
e-st
ress
-ind
uced
apo
ptos
is, w
here
as
Vhh
Cas
p32
resu
lted
in a
popt
osis
. Thes
e V
(H)H
ant
agon
ist a
nd a
goni
st o
f apo
ptos
is c
ould
hav
e po
tent
ial f
or th
e de
velo
pmen
t of
ther
apeu
tics f
or n
euro
dege
nera
tive
dise
ases
and
can
cer,
resp
ectiv
ely.
McG
oni-
gal e
t al.
2009
Prot
ein
kina
se C
Enzy
me
mod
ulat
orTh
e 10
isoz
ymes
of t
he p
rote
in k
inas
e C
(PK
C) f
amily
can
hav
e di
ffere
nt ro
les o
n th
e sa
me
biol
ogic
al p
roce
ss, m
akin
g is
ozym
e sp
ecifi
c an
alys
is o
f fun
ctio
n cr
ucia
l. C
urre
ntly
, onl
y fe
w p
harm
acol
ogic
al c
ompo
unds
with
mod
erat
e is
ozym
e sp
ecifi
c eff
ects
exi
st
thus
ham
peri
ng re
sear
ch in
to in
divi
dual
PK
C is
ozym
es. Th
e an
tigen
bin
ding
regi
ons o
f cam
elid
sing
le c
hain
ant
ibod
ies (
VH
Hs)
co
uld
prov
ide
a so
lutio
n fo
r obt
aini
ng P
KC
isoz
yme
spec
ific
mod
ulat
ors.
In th
e pr
esen
t stu
dy, w
e ha
ve su
cces
sful
ly se
lect
ed a
nd
char
acte
rize
d PK
C e
psilo
n sp
ecifi
c V
HH
ant
ibod
ies f
rom
two
imm
une
VH
H li
brar
ies u
sing
pha
ge d
ispl
ay. Th
e V
HH
s wer
e sh
own
to e
xclu
sive
ly b
ind
to P
KC
eps
ilon
in E
LISA
and
imm
unop
reci
pita
tion
stud
ies.
Str
ikin
gly,
five
of th
e V
HH
s had
an
effec
t on
PKC
eps
ilon
kina
se a
ctiv
ity in
vitr
o. V
HH
s A10
, C1
and
D1
incr
ease
d PK
C e
psilo
n ki
nase
act
ivity
in a
con
cent
ratio
n-de
pend
ent
man
ner (
EC(5
0) v
alue
s: 21
2–31
0 nM
), w
here
as E
6 an
d G
8 in
hibi
ted
PKC
eps
ilon
activ
ity (I
C(5
0) v
alue
s: 10
3–23
3 nM
). N
one
of
thes
e V
HH
s had
an
effec
t on
the
activ
ity o
f the
oth
er n
ovel
PK
C is
ozym
es P
KC
del
ta a
nd P
KC
thet
a. T
o ou
r kno
wle
dge,
thes
e an
tibod
ies a
re th
e fir
st d
escr
ibed
VH
H a
ctiv
ator
s and
inhi
bito
rs fo
r a p
rote
in k
inas
e. F
urth
erm
ore,
the
deve
lopm
ent o
f PK
C e
psi-
lon
spec
ific
mod
ulat
ors i
s an
impo
rtan
t con
trib
utio
n to
PK
C re
sear
ch.
Paal
anen
et
al.
2011
IRF-
1Tu
mor
sup
pres
sor
Mf1
dom
ain
Tran
scri
ptio
nal a
ctiv
ity
IRF-
1 is
a tu
mor
supp
ress
or p
rote
in th
at a
ctiv
ates
gen
e ex
pres
sion
from
a ra
nge
of p
rom
oter
s in
resp
onse
to st
imul
i spa
nnin
g vi
ral
infe
ctio
n to
DN
A d
amag
e. S
tudi
es o
n th
e po
st-t
rans
latio
nal r
egul
atio
n of
IRF-
1 ha
ve b
een
ham
pere
d by
a la
ck o
f sui
tabl
e bi
o-ch
emic
al to
ols c
apab
le o
f tar
getin
g th
e en
doge
nous
pro
tein
. In
this
stud
y, ph
age
disp
lay
tech
nolo
gy w
as u
sed
to d
evel
op a
mon
o-cl
onal
nan
obod
y ta
rget
ing
the
C-t
erm
inal
Mf1
dom
ain
(res
idue
s 301
–325
) of I
RF-1
. Int
race
llula
r exp
ress
ion
of th
e na
nobo
dy
dem
onst
rate
d th
at th
e tr
ansc
ript
iona
l act
ivity
of I
RF-1
is c
onst
rain
ed b
y th
e M
f1 d
omai
n as
nan
obod
y bi
ndin
g ga
ve a
n in
crea
se in
ex
pres
sion
from
IRF-
1-re
spon
sive
pro
mot
ers o
f up
to 8
-fol
d. F
urth
erm
ore,
Mf1
-dir
ecte
d na
nobo
dies
hav
e re
veal
ed a
n un
expe
cted
fu
nctio
n fo
r thi
s dom
ain
in li
miti
ng th
e ra
te a
t whi
ch th
e IR
F-1
prot
ein
is d
egra
ded.
Thus
, the
incr
ease
in IR
F-1
tran
scri
ptio
nal
activ
ity o
bser
ved
on n
anob
ody
bind
ing
is a
ccom
pani
ed b
y a
sign
ifica
nt re
duct
ion
in th
e ha
lf-lif
e of
the
prot
ein.
In su
ppor
t of t
he
data
obt
aine
d us
ing
nano
bodi
es, a
sing
le p
oint
mut
atio
n (P
325A
) inv
olvi
ng th
e C
-ter
min
al re
sidu
e of
IRF-
1 ha
s bee
n id
entifi
ed,
whi
ch re
sults
in g
reat
er tr
ansc
ript
iona
l act
ivity
and
a si
gnifi
cant
incr
ease
in th
e ra
te o
f deg
rada
tion.
The
resu
lts p
rese
nted
her
e su
ppor
t a ro
le fo
r the
Mf1
dom
ain
in li
miti
ng b
oth
IRF-
1-de
pend
ent t
rans
crip
tion
and
the
rate
of I
RF-1
turn
over
. In
addi
tion,
the
data
hig
hlig
ht a
rout
e fo
r act
ivat
ion
of d
owns
trea
m g
enes
in th
e IR
F-1
tum
or su
ppre
ssor
pat
hway
usi
ng b
iolo
gics
.
Mol
ler e
t al
. 201
0
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
483
Hep
atit
is B
HB
eAg
IgN
AR
Intr
acel
lula
r pr
ecor
e pr
otei
n
The
Hep
atiti
s B v
irus
pre
core
pro
tein
is p
roce
ssed
in th
e en
dopl
asm
ic re
ticul
um (E
R) in
to se
cret
ed h
epat
itis B
e a
ntig
en (H
BeA
g),
whi
ch a
cts a
s an
imm
une
tole
roge
n to
est
ablis
h ch
roni
c in
fect
ion.
Dow
nreg
ulat
ion
of se
cret
ed H
BeA
g sh
ould
impr
ove
clin
ical
ou
tcom
e, a
s pat
ient
s who
effe
ctiv
ely
resp
ond
to c
urre
nt tr
eatm
ents
(IFN
-alp
ha) h
ave
sign
ifica
ntly
low
er se
rum
HBe
Ag
leve
ls.
Her
e, w
e de
scri
be a
nov
el re
agen
t, a
sing
le v
aria
ble
dom
ain
(V(N
AR)
) of t
he sh
ark
imm
unog
lobu
lin n
ew a
ntig
en re
cept
or (I
gNA
R)
antib
odie
s. V
(NA
RS) p
osse
ss a
dvan
tage
s in
stab
ility
, siz
e (s
imila
r to
14 k
Da)
and
cry
ptic
epi
tope
reco
gniti
on c
ompa
red
to c
onve
n-tio
nal a
ntib
odie
s. Th
e V
(NA
R) d
omai
n di
spla
yed
biol
ogic
ally
use
ful a
ffini
ty fo
r rec
ombi
nant
and
nat
ive
HBe
Ag,
and
reco
gnis
ed
a un
ique
con
form
atio
nal e
pito
pe. T
o as
sess
ther
apeu
tic p
oten
tial i
n ta
rget
ing
intr
acel
lula
r pre
core
pro
tein
to re
duce
secr
eted
H
BeA
g, th
e V
(NA
R) w
as e
ngin
eere
d fo
r ER-
targ
eted
in v
itro
deliv
ery
to fu
nctio
n as
an
intr
acel
lula
r ant
ibod
y (in
trab
ody)
. In
vitr
o da
ta fr
om H
BV/p
reco
re h
epat
ocyt
e ce
ll lin
es d
emon
stra
ted
effec
tive
intr
abod
y re
gula
tion
of p
reco
re/H
BeA
g.
Wal
sh e
t al
. 201
1
Hep
atit
is B
vir
usH
BcA
gH
BeA
g
Six
sing
le-d
omai
n an
tibod
ies (
VH
Hs)
targ
etin
g th
e co
re a
ntig
en o
f HBV
(HBc
Ag)
hav
e be
en g
ener
ated
and
thre
e of
thes
e bo
und
stro
ngly
to H
BcA
g of
bot
h su
btyp
e ay
w a
nd a
dw. Th
ese
thre
e V
HH
s wer
e st
udie
d as
intr
abod
ies d
irec
ted
tow
ards
the
nucl
eus
or th
e cy
topl
asm
of a
hep
atom
a ce
ll lin
e th
at w
as c
o-tr
ansf
ecte
d w
ith H
BV. A
spec
kled
stai
ning
of H
BcA
g w
as o
bser
ved
in th
e cy
topl
asm
of c
ells
tran
sfec
ted
with
nuc
leot
ropi
c V
HH
intr
abod
ies.
Mor
eove
r, an
incr
ease
d in
trac
ellu
lar a
ccum
ulat
ion
of h
epat
itis
B e
antig
en (H
BeA
g) a
nd a
com
plet
e di
sapp
eara
nce
of in
trac
ellu
lar H
BcA
g si
gnal
wer
e ob
serv
ed w
ith n
ucle
ar ta
rget
ed H
BcA
g-sp
ecifi
c V
HH
s. Th
ese
resu
lts su
gges
t tha
t HBc
Ag-
spec
ific
VH
Hs t
arge
ted
to th
e nu
cleu
s affe
ct H
BcA
g an
d H
BeA
g ex
pres
sion
and
tr
affick
ing
in H
BV-t
rans
fect
ed h
epat
ocyt
es.
Serr
uys e
t al
. 201
0
HIV
-1 v
irus
Rev
pro
tein
Rep
licat
ion
Mul
tim
eriz
atio
n do
mai
n
The
hum
an im
mun
odefi
cien
cy v
irus
, typ
e 1
(HIV
-1)-
enco
ded
Rev
prot
ein
is e
ssen
tial f
or th
e ex
pres
sion
of l
ate
vira
l mRN
As.
Rev
fo
rms a
larg
e or
gani
zed
mul
timer
ic p
rote
in-p
rote
in c
ompl
ex o
n th
e Re
v re
spon
se e
lem
ent o
f the
se v
iral
mRN
A sp
ecie
s and
tran
s-po
rts t
hem
from
the
nucl
eus t
o th
e cy
topl
asm
, exp
loiti
ng th
e C
RM1-
med
iate
d ce
llula
r mac
hine
ry. H
ere
we
repo
rt th
e se
lect
ion
of
a na
nobo
dy, d
eriv
ed fr
om a
llam
a he
avy-
chai
n on
ly a
ntib
ody,
that
effi
cien
tly b
lock
s the
ass
embl
y of
Rev
mul
timer
s. Th
e na
nobo
dy
inhi
bits
HIV
-1 re
plic
atio
n in
cel
ls a
nd sp
ecifi
cally
supp
ress
es th
e Re
v-de
pend
ent e
xpre
ssio
n of
par
tially
splic
ed a
nd u
nspl
iced
H
IV-1
RN
A. I
n H
IV-s
usce
ptib
le c
ells
, thi
s nan
obod
y th
us h
as p
oten
tial a
s an
effec
tive
anti-
HIV
age
nt u
sing
gen
etic
imm
uniz
atio
n st
rate
gies
. Its
bin
ding
site
was
map
ped
to R
ev re
sidu
es L
ys-2
0 an
d Ty
r-23
loca
ted
in th
e N
-ter
min
al a
lpha
-hel
ical
mul
timer
izat
ion
dom
ain.
In th
e pr
esen
ce o
f thi
s nan
obod
y, w
e ob
serv
ed a
n ac
cum
ulat
ion
of d
imer
ic R
ev sp
ecie
s, su
ppor
ting
a he
ad-t
o-he
ad/t
ail-
to-t
ail m
olec
ular
mod
el fo
r Rev
ass
embl
y. Th
e re
sults
indi
cate
that
the
olig
omer
ic a
ssem
bly
of R
ev fo
llow
s an
orde
red
step
wis
e pr
oces
s and
iden
tify
a ne
w e
pito
pe w
ithin
Rev
that
cou
ld g
uide
stra
tegi
es fo
r the
dev
elop
men
t of n
ovel
HIV
inhi
bito
rs.
Ver-
cruy
sse
et
al. 2
010
AD
P-ri
bosy
lati
onSa
lmon
ella
SpvB
toxi
n
AD
P-ri
bosy
latio
n of
hos
t cel
l pro
tein
s is a
com
mon
mod
e of
cel
l int
oxic
atio
n by
pat
hoge
nic
bact
eria
l tox
ins.
Ant
ibod
ies i
nduc
ed
by im
mun
izat
ion
with
inac
tivat
ed A
DP-
ribo
syla
ting
toxi
ns p
rovi
de e
ffici
ent p
rote
ctio
n in
cas
e of
som
e se
cret
ed to
xins
, e.g
., di
ph-
ther
ia a
nd p
ertu
ssis
toxi
ns. H
owev
er, o
ther
AD
P-ri
bosy
latin
g to
xins
, suc
h as
Sal
mon
ella
Spv
B to
xin,
are
secr
eted
dir
ectly
from
the
Salm
onel
la-c
onta
inin
g va
cuol
e in
to th
e cy
toso
l of t
arge
t cel
ls v
ia th
e SP
I-2
enco
ded
bact
eria
l typ
e II
I sec
retio
n sy
stem
, and
thus
ar
e in
acce
ssib
le to
con
vent
iona
l ant
ibod
ies.
Sm
all-m
olec
ule
AD
P-ri
bosy
latio
n in
hibi
tors
are
frau
ght w
ith p
oten
tial s
ide
effec
ts
caus
ed b
y in
hibi
tion
of e
ndog
enou
s AD
P-ri
bosy
ltran
sfer
ases
. Her
e, w
e re
port
the
deve
lopm
ent o
f a si
ngle
-dom
ain
antib
ody
from
an
imm
uniz
ed ll
ama
that
blo
cks t
he c
apac
ity o
f Spv
B to
AD
P-ri
bosy
late
act
in a
t a m
olar
ratio
of 1
: 1.
The
sing
le-d
omai
n an
tibod
y, w
hen
expr
esse
d as
an
intr
abod
y, eff
ectiv
ely
prot
ecte
d ce
lls fr
om th
e cy
toto
xic
activ
ity o
f a tr
ansl
ocat
ion-
com
pete
nt c
him
eric
C
2IN
-C/S
pvB
toxi
n. T
rans
fect
ed c
ells
wer
e al
so p
rote
cted
aga
inst
cyt
oske
leta
l alte
ratio
ns in
duce
d by
wild
-typ
e Sp
vB-e
xpre
ssin
g st
rain
s of S
alm
onel
la. Th
is p
roof
of p
rinc
iple
pav
es th
e w
ay fo
r dev
elop
ing
new
ant
idot
es a
gain
st in
trac
ellu
lar t
oxin
s.
Alz
ogar
ay
et a
l. 20
11
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
484
Bot
ulin
um n
euro
toxi
nsPr
otea
se in
hibi
tor
Botu
linum
neu
roto
xins
(BoN
Ts) f
unct
ion
by d
eliv
erin
g a
prot
ease
to n
euro
nal c
ells
that
cle
ave
SNA
RE p
rote
ins a
nd in
activ
ate
neur
otra
nsm
itter
exo
cyto
sis.
Sm
all (
14 k
Da)
bin
ding
dom
ains
spec
ific
for t
he p
rote
ase
of B
oNT
sero
type
s A o
r B w
ere
sele
cted
fr
om li
brar
ies o
f hea
vy c
hain
onl
y an
tibod
y do
mai
ns (V
HH
s or n
anob
odie
s) c
lone
d fr
om im
mun
ized
alp
acas
. Sev
eral
VH
Hs b
ind
the
BoN
T p
rote
ases
with
hig
h affi
nity
(K(D
) nea
r 1 n
M) a
nd in
clud
e po
tent
inhi
bito
rs o
f BoN
T/A
pro
teas
e ac
tivity
(K(i)
nea
r 1
nM).
The
VH
Hs r
etai
n th
eir b
indi
ng sp
ecifi
city
and
inhi
bito
ry fu
nctio
ns w
hen
expr
esse
d w
ithin
mam
mal
ian
neur
onal
cel
ls a
s in
trab
odie
s. A
VH
H in
hibi
tor o
f BoN
T/A
pro
teas
e w
as a
ble
to p
rote
ct n
euro
nal c
ell S
NA
P25
prot
ein
from
cle
avag
e fo
llow
ing
into
xica
tion
with
BoN
T/A
hol
otox
in. Th
ese
resu
lts d
emon
stra
te th
at V
HH
dom
ains
hav
e po
tent
ial a
s com
pone
nts o
f the
rape
utic
ag
ents
for r
ever
sal o
f bot
ulis
m in
toxi
catio
n.
Trem
blay
et
al.
2010
Intr
acel
lula
r pr
otei
nsE.
col
iIn
trac
ellu
lar
deliv
ery
Mol
ecul
ar s
yrin
ge
Intr
acel
lula
r pro
tein
s hav
e a
grea
t pot
entia
l as t
arge
ts fo
r the
rape
utic
ant
ibod
ies (
Abs
) but
the
plas
ma
mem
bran
e pr
even
ts-
acce
ss to
thes
e an
tigen
s. A
b fr
agm
ents
and
IgG
s are
sele
cted
and
eng
inee
red
in E
. col
i and
this
mic
roor
gani
sm m
ay b
e al
so a
n id
eal v
ecto
r for
thei
r int
race
llula
r del
iver
y. In
this
wor
k w
e de
mon
stra
te th
at si
ngle
-dom
ain
Ab
(sdA
bs) c
an b
e en
gine
ered
to b
e in
ject
ed in
to h
uman
cel
ls b
y E.
col
i bac
teri
a ca
rryi
ng m
olec
ular
syri
nges
ass
embl
ed b
y a
type
III p
rote
in se
cret
ion
syst
em (T
3SS)
. Th
e in
ject
ed sd
Abs
acc
umul
ate
in th
e cy
topl
asm
of H
eLa
cells
at l
evel
s ca.
10(
5)–1
0(6)
mol
ecul
es p
er c
ell a
nd th
eir f
unct
iona
l-ity
is sh
own
by th
e is
olat
ion
of sd
Ab-
antig
en c
ompl
exes
. Inj
ectio
n of
sdA
bs d
oes n
ot re
quir
e ba
cter
ial i
nvas
ion
or th
e tr
ansf
er
of g
enet
ic m
ater
ial.
Thes
e re
sults
are
pro
of-o
f-pr
inci
ple
for t
he c
apac
ity o
f E. c
oli b
acte
ria
to d
irec
tly d
eliv
er in
trac
ellu
lar s
dAbs
(in
trab
odie
s) in
to h
uman
cel
ls fo
r ana
lytic
al a
nd th
erap
eutic
pur
pose
s.
Blan
co-
Tori
bio
et
al. 2
010
Tabl
e 6E
. App
licat
ion
of s
ingl
e-do
mai
n an
tibo
dy fr
agm
ents
in th
erap
y: o
ral a
dmin
istr
atio
n of
sin
gle-
dom
ain
anti
body
frag
men
ts
Ther
mal
sta
bilit
yTr
ypsi
nEx
trem
e pH
Clo
stri
dium
diffi
cile
Toxi
n A
The
extr
eme
pH a
nd p
rote
ase-
rich
env
iron
men
t of t
he u
pper
gas
troi
ntes
tinal
trac
t is a
maj
or o
bsta
cle
faci
ng o
rally
-adm
inis
tere
d pr
otei
n th
erap
eutic
s, in
clud
ing
antib
odie
s. Th
roug
h pr
otei
n en
gine
erin
g, se
vera
l Clo
stri
dium
diffi
cile
toxi
n A
-spe
cific
hea
vy c
hain
an
tibod
y va
riab
le d
omai
ns (V
(H)H
s) w
ere
expr
esse
d w
ith a
n ad
ditio
nal d
isul
fide
bond
by
intr
oduc
ing
Ala
/Gly
54C
ys a
nd Il
e78C
ys
mut
atio
ns. M
utan
t ant
ibod
ies w
ere
com
pare
d to
thei
r wild
-typ
e co
unte
rpar
ts w
ith re
spec
t to
expr
essi
on y
ield
, non
-agg
rega
tion
stat
us, a
ffini
ty fo
r tox
in A
, cir
cula
r dic
hroi
sm (C
D) s
truc
tura
l sig
natu
res,
ther
mal
stab
ility
, pro
teas
e re
sist
ance
, and
toxi
n A
-neu
-tr
aliz
ing
capa
city
. The
mut
ant V
(H)H
s wer
e fo
und
to b
e w
ell e
xpre
ssed
, alth
ough
with
low
er y
ield
s com
pare
d to
wild
-typ
e co
un-
terp
arts
, wer
e no
n-ag
greg
atin
g m
onom
ers,
reta
ined
low
nM
affi
nity
for t
oxin
A, a
lbei
t the
maj
ority
show
ed so
mew
hat r
educ
ed
affini
ty c
ompa
red
to w
ild-t
ype
coun
terp
arts
, and
wer
e ca
pabl
e of
in v
itro
toxi
n A
neu
tral
izat
ion
in c
ell-b
ased
ass
ays.
Far
-UV
and
ne
ar-U
V C
D sp
ectr
osco
py c
onsi
sten
tly sh
owed
shift
s in
peak
inte
nsity
and
sele
ctiv
e pe
ak m
inim
a fo
r wild
-typ
e an
d m
utan
t V(H
)H
pair
s; ho
wev
er, t
he o
vera
ll C
D p
rofil
e re
mai
ned
very
sim
ilar.
A si
gnifi
cant
incr
ease
in th
e th
erm
al u
nfol
ding
mid
poin
t tem
pera
ture
w
as o
bser
ved
for a
ll m
utan
ts a
t bot
h ne
utra
l and
aci
dic
pH. D
iges
tion
of th
e V
(H)H
s with
the
maj
or g
astr
oint
estin
al p
rote
ases
, at
biol
ogic
ally
rele
vant
con
cent
ratio
ns, r
evea
led
a si
gnifi
cant
incr
ease
in p
epsi
n re
sist
ance
for a
ll m
utan
ts a
nd a
n in
crea
se in
chy
mot
-ry
psin
resi
stan
ce fo
r the
maj
ority
of m
utan
ts. M
utan
t V(H
)H tr
ypsi
n re
sist
ance
was
sim
ilar t
o th
at o
f wild
-typ
e V
(H)H
s, a
lthou
gh
the
tryp
sin
resi
stan
ce o
f one
V(H
)H m
utan
t was
sign
ifica
ntly
redu
ced.
Ther
efor
e, th
e in
trod
uctio
n of
a se
cond
dis
ulfid
e bo
nd in
th
e hy
drop
hobi
c co
re n
ot o
nly
incr
ease
s V(H
)H th
erm
al st
abili
ty a
t neu
tral
pH
, as p
revi
ousl
y sh
own,
but
als
o re
pres
ents
a g
ener
ic
stra
tegy
to in
crea
se V
(H)H
stab
ility
at l
ow p
H a
nd im
part
pro
teas
e re
sist
ance
, with
onl
y m
inor
per
turb
atio
ns in
targ
et b
indi
ng
affini
ties.
Thes
e ar
e al
l des
irab
le c
hara
cter
istic
s for
the
desi
gn o
f pro
tein
-bas
ed o
ral t
hera
peut
ics.
Hus
sack
et
al.
2011
a
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
485
Clo
stri
dium
diffi
cile
Nos
ocom
ial i
nfec
tion
Nor
th A
mer
ica
Exot
oxin
A/B
Cel
l rec
epto
r bi
ndin
g do
mai
n
Clo
stri
dium
diffi
cile
is a
lead
ing
caus
e of
nos
ocom
ial i
nfec
tion
in N
orth
Am
eric
a an
d a
cons
ider
able
cha
lleng
e to
hea
lthca
re p
rofe
s-si
onal
s in
hosp
itals
and
nurs
ing
hom
es. Th
e G
ram
-pos
itive
bac
teri
um p
rodu
ces t
wo
high
mol
ecul
ar w
eigh
t exo
toxi
ns, t
oxin
A (T
cdA
) an
d to
xin
B (T
cdB)
, whi
ch a
re th
e m
ajor
vir
ulen
ce fa
ctor
s res
pons
ible
for C
. diffi
cile
-ass
ocia
ted
dise
ase
and
are
targ
ets f
or C
. diffi
cile
-as
soci
ated
dis
ease
ther
apy.
Her
e, re
com
bina
nt si
ngle
-dom
ain
antib
ody
frag
men
ts (V
(H)H
s), w
hich
spec
ifica
lly ta
rget
the
cell
rece
ptor
bi
ndin
g do
mai
ns o
f Tcd
A o
r Tcd
B, w
ere
isol
ated
from
an
imm
une
llam
a ph
age
disp
lay
libra
ry a
nd c
hara
cter
ized
. Fou
r V(H
)Hs (
A4.
2,
A5.
1, A
20.1
, and
A26
.8),
all s
how
n to
reco
gniz
e co
nfor
mat
iona
l epi
tope
s, w
ere
pote
nt n
eutr
aliz
ers o
f the
cyt
opat
hic
effec
ts o
f tox
in
A o
n fib
robl
ast c
ells
in a
n in
vitr
o as
say.
The
neut
raliz
ing
pote
ncy
was
furt
her e
nhan
ced
whe
n V
(H)H
s wer
e ad
min
iste
red
in p
aire
d or
trip
let c
ombi
natio
ns a
t the
sam
e ov
eral
l V(H
)H c
once
ntra
tion,
sugg
estin
g re
cogn
ition
of n
onov
erla
ppin
g Tc
dA e
pito
pes.
Biac
ore
epito
pe m
appi
ng e
xper
imen
ts re
veal
ed th
at so
me
syne
rgis
tic c
ombi
natio
ns c
onsi
sted
of V
(H)H
s rec
ogni
zing
ove
rlapp
ing
epito
pes,
an in
dica
tion
that
fact
ors o
ther
than
mer
e ep
itope
blo
ckin
g ar
e re
spon
sibl
e fo
r the
incr
ease
d ne
utra
lizat
ion.
Fur
ther
bin
ding
ass
ays
reve
aled
Tcd
A-s
peci
fic V
(H)H
s neu
tral
ized
toxi
n A
by
bind
ing
to si
tes o
ther
than
the
carb
ohyd
rate
bin
ding
poc
ket o
f the
toxi
n. W
ith
favo
rabl
e ch
arac
teri
stic
s suc
h as
hig
h pr
oduc
tion
yiel
d, p
oten
t tox
in n
eutr
aliz
atio
n, a
nd in
trin
sic
stab
ility
, the
se V
(H)H
s are
att
ract
ive
syst
emic
ther
apeu
tics b
ut a
re m
ore
so a
s ora
l the
rape
utic
s in
the
dest
abili
zing
env
ironm
ent o
f the
gas
troi
ntes
tinal
trac
t.
Hus
sack
et
al.
2011
b
Infla
mm
ator
y bo
wel
di
seas
eTu
mor
-nec
rosi
s fa
ctor
Lact
ococ
cus
lact
isSe
cret
ion
Infla
mm
ator
y bo
wel
dis
ease
(IBD
) is a
chr
onic
infla
mm
ator
y ga
stro
inte
stin
al d
isor
der.
Syst
emic
trea
tmen
t of I
BD p
atie
nts w
ith
anti-
tum
or n
ecro
sis f
acto
r (T
NF)
-alp
ha a
ntib
odie
s has
pro
ven
to b
e a
high
ly p
rom
isin
g ap
proa
ch, b
ut se
vera
l dra
wba
cks r
emai
n,
incl
udin
g si
de e
ffect
s rel
ated
to sy
stem
ic a
dmin
istr
atio
n an
d hi
gh c
ost o
f tre
atm
ent.
Lact
ococ
cus l
actis
was
eng
inee
red
to se
cret
e m
onov
alen
t and
biv
alen
t mur
ine
(m) T
NF-
neut
raliz
ing
Nan
obod
ies a
s the
rape
utic
pro
tein
s. Th
ese
ther
apeu
tic p
rote
ins a
re d
eriv
ed
from
frag
men
ts o
f hea
vy-c
hain
cam
elid
ant
ibod
ies a
nd a
re m
ore
stab
le th
an c
onve
ntio
nal a
ntib
odie
s. L
. lac
tis-s
ecre
ted
anti-
mT
NF
Nan
obod
ies n
eutr
aliz
ed m
TN
F in
vitr
o. D
aily
ora
l adm
inis
trat
ion
of N
anob
ody-
secr
etin
g L.
lact
is re
sulte
d in
loca
l del
iver
y of
ant
i-m
TN
F N
anob
odie
s at t
he c
olon
and
sign
ifica
ntly
redu
ced
infla
mm
atio
n in
mic
e w
ith d
extr
an su
lfate
sodi
um (D
SS)-
indu
ced
chro
nic
colit
is. I
n ad
ditio
n, th
is a
ppro
ach
was
als
o su
cces
sful
in im
prov
ing
esta
blis
hed
ente
roco
litis
in in
terl
euki
n 10
(IL1
0)(–
/–) m
ice.
Fi
nally
, L. l
actis
-sec
rete
d an
ti-m
TN
F N
anob
odie
s did
not
inte
rfer
e w
ith sy
stem
ic S
alm
onel
la in
fect
ion
in c
oliti
c IL
10(–
/–) m
ice.
In
conc
lusi
on, t
his r
epor
t det
ails
a n
ew th
erap
eutic
app
roac
h fo
r tre
atm
ent o
f chr
onic
col
itis,
invo
lvin
g in
situ
secr
etio
n of
ant
i-m
TN
F N
anob
odie
s by
oral
ly a
dmin
iste
red
L. la
ctis
bac
teri
a. Th
erap
eutic
app
licat
ion
of th
ese
engi
neer
ed b
acte
ria
coul
d ev
entu
ally
lead
to
mor
e eff
ectiv
e an
d sa
fer m
anag
emen
t of I
BD in
hum
ans.
Vand
en-
brou
cke
et a
l. 20
10
F4 fi
mbr
iae
E. c
oli
Dia
rrho
eaPr
oteo
lyti
c st
abili
tyG
astr
ic fl
uid
We
prev
ious
ly d
emon
stra
ted
that
ora
l app
licat
ion
of th
e re
com
bina
nt si
ngle
-dom
ain
antib
ody
frag
men
t (V
HH
) clo
ne K
609,
di
rect
ed a
gain
st E
sche
rich
ia c
oli F
4 fim
bria
e, re
duce
d E.
col
i-ind
uced
dia
rrho
ea in
pig
lets
, but
onl
y at
hig
h V
HH
dos
es. W
e ha
ve
now
show
n th
at a
larg
e po
rtio
n of
the
oral
ly a
pplie
d K
609
VH
H is
pro
teol
ytic
ally
deg
rade
d in
the
stom
ach.
Str
inge
nt se
lect
ion
for p
rote
olyt
ic st
abili
ty id
entifi
ed se
ven
VH
Hs w
ith 7
- to
138-
fold
incr
ease
d st
abili
ty a
fter
in v
itro
incu
batio
n in
gas
tric
flui
d. B
y D
NA
shuffl
ing
we
obta
ined
four
clo
nes w
ith a
furt
her 1
.5- t
o 3-
fold
incr
ease
d in
vitr
o st
abili
ty. Th
ese
VH
Hs d
iffer
ed b
y at
mos
t te
n am
ino
acid
resi
dues
from
eac
h ot
her a
nd K
609
that
wer
e sc
atte
red
over
the
VH
H se
quen
ce a
nd d
id n
ot o
verl
ap w
ith p
redi
cted
pr
otea
se c
leav
age
site
s. Th
e m
ost s
tabl
e cl
one,
K92
2, re
tain
ed 4
1% a
ctiv
ity a
fter
incu
batio
n in
gas
tric
flui
d an
d 90
% in
jeju
nal fl
uid.
O
ral a
pplic
atio
n of
K92
2 to
pig
lets
con
firm
ed it
s im
prov
ed p
rote
olyt
ic st
abili
ty. I
n ad
ditio
n, K
922
boun
d to
F4
fimbr
iae
with
hig
her
affini
ty a
nd in
hibi
ted
fimbr
ial a
dhes
ion
at lo
wer
VH
H c
once
ntra
tions
. K92
2 is
thus
a p
rom
isin
g ca
ndid
ate
for p
reve
ntio
n of
pig
let
diar
rhoe
a. F
urth
erm
ore,
our
find
ings
cou
ld g
uide
sele
ctio
n an
d im
prov
emen
t by
gene
tic e
ngin
eeri
ng o
f oth
er re
com
bina
nt a
nti-
body
frag
men
ts fo
r ora
l use
.
Har
m-
sen
et a
l. 20
06
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
486
Rot
avir
usD
iarr
hoea
Dev
elop
ing
coun
trie
sPa
ssiv
e im
mun
othe
rapy
Tran
sfor
med
lact
obac
illi
Back
grou
nd. R
otav
irus
-indu
ced
diar
rhea
pos
es a
wor
ldw
ide
med
ical
pro
blem
in c
ausi
ng su
bsta
ntia
l mor
bidi
ty a
nd m
orta
lity
amon
g ch
ildre
n in
dev
elop
ing
coun
trie
s. W
e th
eref
ore
deve
lope
d a
syst
em fo
r pas
sive
imm
unot
hera
py in
whi
ch re
com
bina
nt
lact
obac
illi c
onst
itutiv
ely
expr
ess n
eutr
aliz
ing
vari
able
dom
ain
of ll
ama
heav
y-ch
ain
(VH
H) a
ntib
ody
frag
men
ts a
gain
st ro
tavi
rus.
Met
hods
. VH
H w
ere
expr
esse
d in
Lac
toba
cillu
s par
acas
ei, i
n bo
th se
cret
ed a
nd c
ell s
urfa
ce-a
ncho
red
form
s. E
lect
ron
mic
rosc
opy
was
use
d to
inve
stig
ate
the
bind
ing
effica
cy o
f VH
H-e
xpre
ssin
g la
ctob
acill
i. To
inve
stig
ate
the
in v
ivo
func
tion
of V
HH
-exp
ress
ing
lact
obac
illi,
a m
ouse
pup
mod
el o
f rot
avir
us in
fect
ion
was
use
d. R
esul
ts. E
ffici
ent b
indi
ng o
f the
VH
H a
ntib
ody
frag
men
ts to
ro
tavi
rus w
as sh
own
by e
nzym
e-lin
ked
imm
unos
orbe
nt a
ssay
and
scan
ning
ele
ctro
n m
icro
scop
y. V
HH
frag
men
ts e
xpre
ssed
by
lact
obac
illi c
onfe
rred
a si
gnifi
cant
redu
ctio
n in
infe
ctio
n in
cel
l cul
ture
s. W
hen
adm
inis
tere
d or
ally
, lac
toba
cilli
-pro
duci
ng su
rfac
e-ex
pres
sed
VH
H m
arke
dly
shor
tene
d di
seas
e du
ratio
n, se
veri
ty, a
nd v
iral
load
in a
mou
se m
odel
of r
otav
irus
-ind
uced
dia
rrhe
a w
hen
adm
inis
tere
d bo
th fr
esh
and
in a
free
ze-d
ried
form
. Con
clus
ions
. Tra
nsfo
rmed
lact
obac
illi m
ay fo
rm th
e ba
sis o
f a n
ovel
form
of
prop
hyla
ctic
trea
tmen
t aga
inst
rota
viru
s inf
ectio
ns a
nd o
ther
dia
rrhe
al d
isea
ses.
Pant
et
al. 2
006
Tabl
e 6F
. App
licat
ion
of s
ingl
e-do
mai
n an
tibo
dy fr
agm
ents
in th
erap
y: p
reve
ntio
n of
am
yloi
d pl
aque
form
atio
n an
d pr
otei
n ag
greg
atio
n
Bet
a-am
yloi
d tr
ansm
i-gr
atio
n effi
cien
cyA
lzhe
imer
’s di
seas
eC
ereb
ral a
myl
oid
an
giop
athy
Prev
ious
ly se
lect
ed a
myl
oid
beta
reco
gniz
ing
heav
y ch
ain
antib
ody
frag
men
ts (V
HH
) affi
nity
bin
ders
der
ived
from
the
Cam
e lid
hea
vy
chai
n an
tibod
y re
pert
oire
wer
e te
sted
for t
heir
prop
ensi
ty to
cro
ss th
e bl
ood-
brai
n ba
rrie
r (BB
B) u
sing
an
esta
blis
hed
in v
itro
BBB
co-
cultu
re sy
stem
. Of a
ll te
sted
VH
H, n
i3A
show
ed h
ighe
st tr
ansm
igra
tion
effici
ency
whi
ch is
, in
part
, fac
ilita
ted
by a
thre
e am
ino
acid
su
bstit
utio
ns in
its N
-ter
min
al d
omai
n. A
dditi
onal
stud
ies i
ndic
ated
that
the
mec
hani
sm o
f tra
nsce
llula
r pas
sage
of n
i3A
is b
y ac
tive
tran
spor
t. A
s VH
H n
i3A
com
bine
s the
abi
lity
to re
cogn
ize
amyl
oid
beta
and
to c
ross
the
BBB,
it h
as p
oten
tial a
s a to
ol fo
r non
-inva
-si
ve in
viv
o im
agin
g an
d as
effi
cien
t loc
al d
rug
targ
etin
g m
oiet
y in
pat
ient
s suff
erin
g fr
om c
ereb
ral a
myl
oido
sis s
uch
as A
lzhe
imer
’s di
seas
e (A
D) a
nd c
ereb
ral a
myl
oid
angi
opat
hy (C
AA
).
Rutg
ers
et a
l. 20
11
Bet
a-am
yloi
dA
lzhe
imer
’s di
seas
ePr
oteo
lyti
c na
nobo
dyA
ggre
gati
on
Dep
ositi
on o
f bet
a-am
yloi
d (A
bet
a) is
con
side
red
an im
port
ant e
arly
eve
nt in
the
path
ogen
esis
of A
lzhe
imer
’s di
seas
e (A
D),
and
redu
ctio
n of
A b
eta
leve
ls in
the
brai
n co
uld
be a
via
ble
ther
apeu
tic a
ppro
ach.
A p
oten
tially
non
infla
mm
ator
y ro
ute
to fa
cilit
ate
clea
r-an
ce a
nd re
duce
toxi
city
of A
bet
a is
to d
egra
de th
e pe
ptid
e us
ing
prot
eoly
tic n
anob
odie
s. H
ere
we
show
that
a p
rote
olyt
ic n
anob
ody
engi
neer
ed to
cle
ave
A b
eta
at it
s alp
ha-s
ecre
tase
site
has
pot
entia
l the
rape
utic
val
ue. Th
e A
sec-
1A p
rote
olyt
ic n
anob
ody,
deri
ved
from
a p
aren
t cat
alyt
ic li
ght c
hain
ant
ibod
y, pr
even
ts a
ggre
gatio
n of
mon
omer
ic A
bet
a, in
hibi
ts fu
rthe
r agg
rega
tion
of p
refo
rmed
A
beta
agg
rega
tes,
and
redu
ces A
bet
a-in
duce
d cy
toto
xici
ty to
war
d a
hum
an n
euro
blas
tom
a ce
ll lin
e. Th
e na
nobo
dy a
lso
redu
ces t
oxic
-ity
indu
ced
by o
vere
xpre
ssio
n of
the
hum
an a
myl
oid
prec
urso
r pro
tein
(APP
) in
a C
hine
se h
amst
er o
vary
(CH
O) c
ell l
ine
by c
leav
ing
APP
at t
he a
lpha
-sec
reta
se si
te w
hich
pre
clud
es fo
rmat
ion
of A
bet
a. T
arge
ted
prot
eoly
sis o
f APP
and
A b
eta
with
cat
alyt
ic n
anob
od-
ies r
epre
sent
s a n
ovel
ther
apeu
tic a
ppro
ach
for t
reat
ing
AD
whe
re p
oten
tially
har
mfu
l sid
e eff
ects
can
he
min
imiz
ed.
Kas
-tu
rira
n-ga
n et
al.
2010
Am
yloi
doge
nic
lyso
zym
eA
lpha
-syn
ucle
inPa
rkin
son’
s di
seas
eSe
lf-a
ssoc
iati
on
Nan
obod
ies a
re si
ngle
cha
in a
ntib
odie
s tha
t are
uni
quel
y pr
oduc
ed in
Cam
elid
ae, e
.g. c
amel
s and
llam
as. Th
ey h
ave
the
desi
r-ab
le fe
atur
es o
f sm
all s
izes
(Mw
< 1
4 kD
a) a
nd h
igh
affini
ties a
gain
st a
ntig
ens (
Kd
sim
ilar t
o nM
), m
akin
g th
em id
eal a
s str
uctu
ral
prob
es fo
r bio
med
ical
ly re
leva
nt m
otifs
bot
h in
vitr
o an
d in
viv
o. W
e ha
ve p
revi
ousl
y sh
own
that
nan
obod
y bi
ndin
g to
am
yloi
do-
geni
c hu
man
lyso
zym
e va
rian
ts c
an e
ffect
ivel
y in
hibi
t the
ir a
ggre
gatio
n, th
e pr
oces
s tha
t is a
t the
ori
gin
of sy
stem
ic a
myl
oid
dis-
ease
. Her
e w
e re
port
the
NM
R as
sign
men
ts o
f a n
ew n
anob
ody,
term
ed N
bSyn
2, w
hich
reco
gnis
es th
e C
-ter
min
us o
f the
intr
insi
-ca
lly d
isor
dere
d pr
otei
n, h
uman
alp
ha-s
ynuc
lein
(aS)
, who
se a
berr
ant s
elf-
asso
ciat
ion
is im
plic
ated
in P
arki
nson
’s di
seas
e.
Vuch
elen
et
al.
2009
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
487
Rot
avir
usD
iarr
hoea
Dev
elop
ing
coun
trie
sPa
ssiv
e im
mun
othe
rapy
Tran
sfor
med
lact
obac
illi
Back
grou
nd. R
otav
irus
-indu
ced
diar
rhea
pos
es a
wor
ldw
ide
med
ical
pro
blem
in c
ausi
ng su
bsta
ntia
l mor
bidi
ty a
nd m
orta
lity
amon
g ch
ildre
n in
dev
elop
ing
coun
trie
s. W
e th
eref
ore
deve
lope
d a
syst
em fo
r pas
sive
imm
unot
hera
py in
whi
ch re
com
bina
nt
lact
obac
illi c
onst
itutiv
ely
expr
ess n
eutr
aliz
ing
vari
able
dom
ain
of ll
ama
heav
y-ch
ain
(VH
H) a
ntib
ody
frag
men
ts a
gain
st ro
tavi
rus.
Met
hods
. VH
H w
ere
expr
esse
d in
Lac
toba
cillu
s par
acas
ei, i
n bo
th se
cret
ed a
nd c
ell s
urfa
ce-a
ncho
red
form
s. E
lect
ron
mic
rosc
opy
was
use
d to
inve
stig
ate
the
bind
ing
effica
cy o
f VH
H-e
xpre
ssin
g la
ctob
acill
i. To
inve
stig
ate
the
in v
ivo
func
tion
of V
HH
-exp
ress
ing
lact
obac
illi,
a m
ouse
pup
mod
el o
f rot
avir
us in
fect
ion
was
use
d. R
esul
ts. E
ffici
ent b
indi
ng o
f the
VH
H a
ntib
ody
frag
men
ts to
ro
tavi
rus w
as sh
own
by e
nzym
e-lin
ked
imm
unos
orbe
nt a
ssay
and
scan
ning
ele
ctro
n m
icro
scop
y. V
HH
frag
men
ts e
xpre
ssed
by
lact
obac
illi c
onfe
rred
a si
gnifi
cant
redu
ctio
n in
infe
ctio
n in
cel
l cul
ture
s. W
hen
adm
inis
tere
d or
ally
, lac
toba
cilli
-pro
duci
ng su
rfac
e-ex
pres
sed
VH
H m
arke
dly
shor
tene
d di
seas
e du
ratio
n, se
veri
ty, a
nd v
iral
load
in a
mou
se m
odel
of r
otav
irus
-ind
uced
dia
rrhe
a w
hen
adm
inis
tere
d bo
th fr
esh
and
in a
free
ze-d
ried
form
. Con
clus
ions
. Tra
nsfo
rmed
lact
obac
illi m
ay fo
rm th
e ba
sis o
f a n
ovel
form
of
prop
hyla
ctic
trea
tmen
t aga
inst
rota
viru
s inf
ectio
ns a
nd o
ther
dia
rrhe
al d
isea
ses.
Pant
et
al. 2
006
Tabl
e 6F
. App
licat
ion
of s
ingl
e-do
mai
n an
tibo
dy fr
agm
ents
in th
erap
y: p
reve
ntio
n of
am
yloi
d pl
aque
form
atio
n an
d pr
otei
n ag
greg
atio
n
Bet
a-am
yloi
d tr
ansm
i-gr
atio
n effi
cien
cyA
lzhe
imer
’s di
seas
eC
ereb
ral a
myl
oid
an
giop
athy
Prev
ious
ly se
lect
ed a
myl
oid
beta
reco
gniz
ing
heav
y ch
ain
antib
ody
frag
men
ts (V
HH
) affi
nity
bin
ders
der
ived
from
the
Cam
e lid
hea
vy
chai
n an
tibod
y re
pert
oire
wer
e te
sted
for t
heir
prop
ensi
ty to
cro
ss th
e bl
ood-
brai
n ba
rrie
r (BB
B) u
sing
an
esta
blis
hed
in v
itro
BBB
co-
cultu
re sy
stem
. Of a
ll te
sted
VH
H, n
i3A
show
ed h
ighe
st tr
ansm
igra
tion
effici
ency
whi
ch is
, in
part
, fac
ilita
ted
by a
thre
e am
ino
acid
su
bstit
utio
ns in
its N
-ter
min
al d
omai
n. A
dditi
onal
stud
ies i
ndic
ated
that
the
mec
hani
sm o
f tra
nsce
llula
r pas
sage
of n
i3A
is b
y ac
tive
tran
spor
t. A
s VH
H n
i3A
com
bine
s the
abi
lity
to re
cogn
ize
amyl
oid
beta
and
to c
ross
the
BBB,
it h
as p
oten
tial a
s a to
ol fo
r non
-inva
-si
ve in
viv
o im
agin
g an
d as
effi
cien
t loc
al d
rug
targ
etin
g m
oiet
y in
pat
ient
s suff
erin
g fr
om c
ereb
ral a
myl
oido
sis s
uch
as A
lzhe
imer
’s di
seas
e (A
D) a
nd c
ereb
ral a
myl
oid
angi
opat
hy (C
AA
).
Rutg
ers
et a
l. 20
11
Bet
a-am
yloi
dA
lzhe
imer
’s di
seas
ePr
oteo
lyti
c na
nobo
dyA
ggre
gati
on
Dep
ositi
on o
f bet
a-am
yloi
d (A
bet
a) is
con
side
red
an im
port
ant e
arly
eve
nt in
the
path
ogen
esis
of A
lzhe
imer
’s di
seas
e (A
D),
and
redu
ctio
n of
A b
eta
leve
ls in
the
brai
n co
uld
be a
via
ble
ther
apeu
tic a
ppro
ach.
A p
oten
tially
non
infla
mm
ator
y ro
ute
to fa
cilit
ate
clea
r-an
ce a
nd re
duce
toxi
city
of A
bet
a is
to d
egra
de th
e pe
ptid
e us
ing
prot
eoly
tic n
anob
odie
s. H
ere
we
show
that
a p
rote
olyt
ic n
anob
ody
engi
neer
ed to
cle
ave
A b
eta
at it
s alp
ha-s
ecre
tase
site
has
pot
entia
l the
rape
utic
val
ue. Th
e A
sec-
1A p
rote
olyt
ic n
anob
ody,
deri
ved
from
a p
aren
t cat
alyt
ic li
ght c
hain
ant
ibod
y, pr
even
ts a
ggre
gatio
n of
mon
omer
ic A
bet
a, in
hibi
ts fu
rthe
r agg
rega
tion
of p
refo
rmed
A
beta
agg
rega
tes,
and
redu
ces A
bet
a-in
duce
d cy
toto
xici
ty to
war
d a
hum
an n
euro
blas
tom
a ce
ll lin
e. Th
e na
nobo
dy a
lso
redu
ces t
oxic
-ity
indu
ced
by o
vere
xpre
ssio
n of
the
hum
an a
myl
oid
prec
urso
r pro
tein
(APP
) in
a C
hine
se h
amst
er o
vary
(CH
O) c
ell l
ine
by c
leav
ing
APP
at t
he a
lpha
-sec
reta
se si
te w
hich
pre
clud
es fo
rmat
ion
of A
bet
a. T
arge
ted
prot
eoly
sis o
f APP
and
A b
eta
with
cat
alyt
ic n
anob
od-
ies r
epre
sent
s a n
ovel
ther
apeu
tic a
ppro
ach
for t
reat
ing
AD
whe
re p
oten
tially
har
mfu
l sid
e eff
ects
can
he
min
imiz
ed.
Kas
-tu
rira
n-ga
n et
al.
2010
Am
yloi
doge
nic
lyso
zym
eA
lpha
-syn
ucle
inPa
rkin
son’
s di
seas
eSe
lf-a
ssoc
iati
on
Nan
obod
ies a
re si
ngle
cha
in a
ntib
odie
s tha
t are
uni
quel
y pr
oduc
ed in
Cam
elid
ae, e
.g. c
amel
s and
llam
as. Th
ey h
ave
the
desi
r-ab
le fe
atur
es o
f sm
all s
izes
(Mw
< 1
4 kD
a) a
nd h
igh
affini
ties a
gain
st a
ntig
ens (
Kd
sim
ilar t
o nM
), m
akin
g th
em id
eal a
s str
uctu
ral
prob
es fo
r bio
med
ical
ly re
leva
nt m
otifs
bot
h in
vitr
o an
d in
viv
o. W
e ha
ve p
revi
ousl
y sh
own
that
nan
obod
y bi
ndin
g to
am
yloi
do-
geni
c hu
man
lyso
zym
e va
rian
ts c
an e
ffect
ivel
y in
hibi
t the
ir a
ggre
gatio
n, th
e pr
oces
s tha
t is a
t the
ori
gin
of sy
stem
ic a
myl
oid
dis-
ease
. Her
e w
e re
port
the
NM
R as
sign
men
ts o
f a n
ew n
anob
ody,
term
ed N
bSyn
2, w
hich
reco
gnis
es th
e C
-ter
min
us o
f the
intr
insi
-ca
lly d
isor
dere
d pr
otei
n, h
uman
alp
ha-s
ynuc
lein
(aS)
, who
se a
berr
ant s
elf-
asso
ciat
ion
is im
plic
ated
in P
arki
nson
’s di
seas
e.
Vuch
elen
et
al.
2009
Agg
rega
tion
Alp
ha-s
ynuc
lein
Fibr
illar
am
yloi
d
stru
ctur
esPa
rkin
son’
s di
seas
e
The
aggr
egat
ion
of th
e in
trin
sica
lly d
isor
dere
d pr
otei
n al
pha-
synu
clei
n to
form
fibr
illar
am
yloi
d st
ruct
ures
is in
timat
ely
asso
ciat
ed
with
a v
arie
ty o
f neu
rolo
gica
l dis
orde
rs, m
ost n
otab
ly P
arki
nson
’s di
seas
e. Th
e m
olec
ular
mec
hani
sm o
f alp
ha-s
ynuc
lein
agg
rega
-tio
n an
d to
xici
ty is
not
yet
und
erst
ood
in a
ny d
etai
l, no
t lea
st b
ecau
se o
f the
pau
city
of s
truc
tura
l pro
bes t
hrou
gh w
hich
to st
udy
the
beha
vior
of s
uch
a di
sord
ered
syst
em. H
ere,
we
desc
ribe
an
inve
stig
atio
n in
volv
ing
a si
ngle
-dom
ain
cam
elid
ant
ibod
y, N
bSyn
2,
sele
cted
by
phag
e di
spla
y te
chni
ques
to b
ind
to a
lpha
-syn
ucle
in, i
nclu
ding
the
expl
orat
ion
of it
s effe
cts o
n th
e in
vitr
o ag
greg
atio
n of
th
e pr
otei
n un
der a
var
iety
of c
ondi
tions
. We
show
usi
ng is
othe
rmal
cal
orim
etri
c m
etho
ds th
at N
bSyn
2 bi
nds s
peci
fical
ly to
mon
o-m
eric
alp
ha-s
ynuc
lein
with
nan
omol
ar a
ffini
ty a
nd b
y m
eans
of N
MR
spec
tros
copy
that
it in
tera
cts w
ith th
e fo
ur C
-ter
min
al re
sidu
es
of th
e pr
otei
n. Th
is la
tter fi
ndin
g is
con
firm
ed b
y th
e de
term
inat
ion
of a
cry
stal
stru
ctur
e of
NbS
yn2
boun
d to
a p
eptid
e en
com
pass
ing
the
nine
C-t
erm
inal
resi
dues
of a
lpha
-syn
ucle
in. Th
e N
bSyn
2 : a
lpha
-syn
ucle
in in
tera
ctio
n is
med
iate
d m
ainl
y by
side
-cha
in in
tera
c-tio
ns w
hile
wat
er m
olec
ules
cro
ss-li
nk th
e m
ain-
chai
n at
oms o
f alp
ha-s
ynuc
lein
to a
tom
s of N
bSyn
2, a
feat
ure
we
belie
ve c
ould
be
impo
rtan
t in
intr
insi
cally
dis
orde
red
prot
ein
inte
ract
ions
mor
e ge
nera
lly. Th
e ag
greg
atio
n be
havi
or o
f alp
ha-s
ynuc
lein
at p
hysi
olog
ical
pH
, inc
ludi
ng th
e m
orph
olog
y of
the
resu
lting
fibr
illar
stru
ctur
es, i
s rem
arka
bly
unaff
ecte
d by
the
pres
ence
of N
bSyn
2 an
d in
deed
we
show
that
NbS
yn2
bind
s str
ongl
y to
the
aggr
egat
ed a
s wel
l as t
o th
e so
lubl
e fo
rms o
f alp
ha-s
ynuc
lein
. Thes
e re
sults
giv
e st
rong
sup-
port
to th
e co
njec
ture
that
the
C-t
erm
inal
regi
on o
f the
pro
tein
is n
ot d
irect
ly in
volv
ed in
the
mec
hani
sm o
f agg
rega
tion
and
sugg
est
that
bin
ding
of N
bSyn
2 co
uld
be a
use
ful p
robe
for t
he id
entifi
catio
n of
alp
ha-s
ynuc
lein
agg
rega
tion
in v
itro
and
poss
ibly
in v
ivo.
De
Gen
st
et a
l. 20
10
Agg
rega
tion
Ocu
loph
aryn
geal
m
uscu
lar
dyst
roph
yA
lani
ne e
xpan
sion
PAB
PN1
Ocu
loph
aryn
geal
mus
cula
r dys
trop
hy is
cau
sed
by sm
all a
lani
ne e
xpan
sion
s in
poly
aden
ylat
e bi
ndin
g pr
otei
n nu
clea
r 1 (P
ABP
N1)
pr
otei
n re
sulti
ng in
its i
ntra
nucl
ear a
ccum
ulat
ion
in sk
elet
al m
uscl
e. 3
F5 ll
ama
antib
ody
spec
ifica
lly in
terf
eres
with
the
PABP
N1
aggr
egat
ion
proc
ess i
n vi
tro
and
in v
ivo.
To
unde
rsta
nd th
e st
ruct
ural
bas
is fo
r its
epi
tope
reco
gniti
on w
e m
appe
d th
e bi
ndin
g in
terf
ace
of 3
F5 w
ith P
ABP
N1
and
prov
ide
a st
ruct
ural
mod
el o
f the
3F5
-PA
BPN
1 co
mpl
ex. W
e sh
ow th
at 3
F5 c
ompl
emen
tari
ty
dete
rmin
ing
regi
ons c
reat
e a
cavi
ty in
whi
ch P
ABP
N1
alph
a-he
lix d
omai
n re
side
s by
invo
lvin
g cr
itica
l res
idue
s pre
viou
sly
impl
i-ca
ted
in th
e ag
greg
atio
n pr
oces
s. Th
ese
resu
lts m
ay in
crea
se o
ur u
nder
stan
ding
of t
he P
ABP
N1
aggr
egat
ion
mec
hani
sm a
nd th
e th
erap
eutic
pot
entia
l of 3
F5.
Impa
gli-
azzo
et
al. 2
010
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
488
Tabl
e 6G
. App
licat
ion
of s
ingl
e-do
mai
n an
tibo
dy fr
agm
ents
in th
erap
y: m
ulti
spec
ific
and
mul
tifu
ncti
onal
con
stru
cts
Pent
abod
y Ve
roto
xin
B-s
ubun
itE.
col
iTh
erm
osta
bilit
yPr
otea
se r
esis
tanc
e
We
desc
ribe
a n
ovel
type
of m
olec
ule
in w
hich
sing
le-d
omai
n an
tibod
ies (
sdA
bs) i
sola
ted
from
a n
aive
llam
a si
ngle
dom
ain
anti-
body
libr
ary
are
linke
d to
an
olig
omer
izat
ion
dom
ain
to g
ener
ate
high
-avi
dity
ant
igen
-bin
ding
reag
ents
. An
sdA
b is
fuse
d to
the
B-su
buni
t of E
sche
rich
ia c
oli v
erot
oxin
, or s
higa
-like
toxi
n, w
hich
self-
asse
mbl
es to
form
a h
omop
enta
mer
and
resu
lts in
sim
ul-
tane
ous s
dAb
pent
amer
izat
ion
and
intr
oduc
tion
of a
vidi
ty. M
olec
ular
mod
elin
g in
dica
ted
that
this
fusi
on p
rote
in (P
DB:
10J
F),
term
ed p
enta
body
, has
stru
ctur
al fl
exib
ility
for b
indi
ng to
surf
ace-
pres
ente
d an
tigen
. In
the
inst
ance
of a
n sd
Ab
spec
ific
for a
pe
ptid
e an
tigen
, pen
tam
eriz
atio
n re
sulte
d in
a d
ram
atic
incr
ease
in fu
nctio
nal a
ffini
ty fo
r im
mob
ilize
d an
tigen
. The
pent
abod
y w
as
expr
esse
d in
hig
h yi
eld
in E
. col
i in
a no
n-ag
greg
ated
stat
e, a
nd e
xhib
ited
exce
llent
ther
mos
tabi
lity
and
prot
ease
resi
stan
ce. Th
is
tech
nolo
gy p
rovi
des a
rela
tivel
y ra
pid
mea
ns o
f gen
erat
ing
nove
l ant
igen
-bin
ding
mol
ecul
es th
at b
ind
stro
ngly
to im
mob
ilize
d an
ti-ge
n. It
is e
xpec
ted
that
pen
tava
lent
sdA
bs w
ill h
ave
gene
ral a
pplic
abili
ty in
pro
teom
ics,
imm
unoc
hem
ical
stai
ning
, can
cer d
iagn
osis
an
d ot
her a
pplic
atio
ns in
whi
ch a
ntig
ens a
re p
rese
nted
mul
tival
ently
.
Zha
ng e
t al
. 200
4
Bip
arat
opic
nan
obod
yEp
ider
mal
gro
wth
fact
or
rece
ptor
Ant
ican
cer
ther
apeu
tics
The
epid
erm
al g
row
th fa
ctor
rece
ptor
(EG
FR) h
as b
een
show
n to
be
a va
lid c
ance
r tar
get f
or a
ntib
ody-
base
d th
erap
y. A
t pre
sent
, se
vera
l ant
i-EG
FR m
onoc
lona
l ant
ibod
ies h
ave
been
succ
essf
ully
use
d, su
ch a
s cet
uxim
ab a
nd m
atuz
umab
. X-r
ay c
ryst
allo
grap
hy
data
show
that
thes
e an
tibod
ies b
ind
to d
iffer
ent e
pito
pes o
n th
e ec
to-d
omai
n of
EG
FR, p
rovi
ding
a ra
tiona
le fo
r the
com
bine
d us
e of
thes
e tw
o an
tibod
y sp
ecifi
citie
s. W
e ha
ve p
revi
ousl
y re
port
ed o
n th
e su
cces
sful
isol
atio
n of
ant
agon
istic
ant
i-EG
FR n
anob
od-
ies.
In o
ur st
udy,
we
aim
ed to
impr
ove
the
effica
cy o
f the
se m
olec
ules
by
com
bini
ng n
anob
odie
s with
spec
ifici
ties s
imila
r to
both
ce
tuxi
mab
and
mat
uzum
ab in
to a
sing
le b
ipar
atop
ic m
olec
ule.
Car
eful
ly d
esig
ned
phag
e na
nobo
dy se
lect
ions
resu
lted
in tw
o se
ts
of n
anob
odie
s tha
t spe
cific
ally
blo
cked
the
bind
ing
of e
ither
mat
uzum
ab o
r cet
uxim
ab to
EG
FR a
nd th
at d
id n
ot c
ompe
te fo
r eac
h ot
hers
’ bin
ding
. A c
ombi
natio
n of
nan
obod
ies f
rom
bot
h ep
itope
gro
ups i
nto
the
bipa
rato
pic
nano
body
CO
NA
N-1
was
show
n to
bl
ock
EGFR
act
ivat
ion
mor
e effi
cien
tly th
an m
onov
alen
t or b
ival
ent (
mon
ospe
cific
) nan
obod
ies.
In a
dditi
on, t
his b
ipar
atop
ic n
ano-
body
pot
ently
inhi
bite
d EG
F-de
pend
ent c
ell p
rolif
erat
ion.
Impo
rtan
tly, i
n an
in v
ivo
mod
el o
f ath
ymic
mic
e be
arin
g A
431
xeno
-gr
afts
, CO
NA
N-1
inhi
bite
d tu
mou
r out
grow
th w
ith a
n al
mos
t sim
ilar p
oten
cy a
s the
who
le m
Ab
cetu
xim
ab, d
espi
te th
e fa
ct th
at
CO
NA
N-1
is d
evoi
d of
an
Fc p
ortio
n th
at c
ould
med
iate
imm
une
effec
tor f
unct
ions
. Com
pare
d to
ther
apy
usin
g bi
vale
nt, m
ono-
spec
ific
nano
bodi
es, C
ON
AN
-1 w
as c
lear
ly m
ore
pote
nt in
tum
our g
row
th in
hibi
tion.
Thes
e re
sults
show
that
the
ratio
nal d
esig
n of
bip
arat
opic
nan
obod
y-ba
sed
antic
ance
r the
rape
utic
s may
yie
ld p
oten
t lea
d m
olec
ules
for f
urth
er d
evel
opm
ent.
Roov
ers
et a
l. 20
11
Biv
alen
t nan
obod
yIn
fluen
za v
irus
Intr
anas
al a
dmin
istr
a-ti
on
Influ
enza
A v
irus
infe
ctio
ns im
pose
a re
curr
ent a
nd g
loba
l dis
ease
bur
den.
Cur
rent
ant
ivir
als a
gain
st in
fluen
za a
re n
ot a
lway
s eff
ectiv
e. W
e as
sess
ed th
e pr
otec
tive
pote
ntia
l of m
onov
alen
t and
biv
alen
t Nan
obod
ies (
Abl
ynx)
aga
inst
cha
lleng
e w
ith th
is v
irus
. Th
ese
Nan
obod
ies w
ere
deri
ved
from
llam
as a
nd ta
rget
H5N
1 he
mag
glut
inin
. Int
rana
sal a
dmin
istr
atio
n of
Nan
obod
ies e
ffect
ivel
y co
ntro
lled
hom
olog
ous i
nflue
nza
A v
irus
repl
icat
ion.
Adm
inis
trat
ion
of N
anob
odie
s bef
ore
chal
leng
e st
rong
ly re
duce
d H
5N1
viru
s re
plic
atio
n in
the
lung
s and
pro
tect
ed m
ice
from
mor
bidi
ty a
nd m
orta
lity
afte
r a le
thal
cha
lleng
e w
ith H
5N1
viru
s. Th
e bi
vale
nt
Nan
obod
y w
as a
t lea
st 6
0-fo
ld m
ore
effec
tive
than
the
mon
oval
ent N
anob
ody
in c
ontr
ollin
g vi
rus r
eplic
atio
n. In
add
ition
, Nan
o-bo
dy th
erap
y af
ter c
halle
nge
stro
ngly
redu
ced
vira
l rep
licat
ion
and
sign
ifica
ntly
del
ayed
tim
e to
dea
th. E
pito
pe m
appi
ng re
veal
ed
that
the
VH
H N
anob
ody
bind
s to
antig
enic
site
B in
H5
hem
aggl
utin
in. B
ecau
se N
anob
odie
s are
smal
l, st
able
, and
sim
ple
to p
ro-
duce
, the
y ar
e a
prom
isin
g, n
ovel
ther
apeu
tic a
gent
aga
inst
influ
enza
.
Iban
ez e
t al
. 201
1
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
489
Mul
tim
eric
and
bi
spec
ific
cons
truc
tsIn
fluen
za
For e
ffici
ent p
reve
ntio
n of
vir
al in
fect
ions
and
cro
ss p
rote
ctio
n, si
mul
tane
ous t
arge
ting
of m
ultip
le v
iral
epi
tope
s is a
pow
er-
ful s
trat
egy.
Llam
a he
avy
chai
n an
tibod
y fr
agm
ents
(VH
H) a
gain
st th
e tr
imer
ic e
nvel
ope
prot
eins
of R
espi
rato
ry S
yncy
tial V
irus
(F
usio
n pr
otei
n), R
abie
s vir
us (G
lyco
prot
ein)
and
H5N
1 In
fluen
za (H
emag
glut
inin
5) w
ere
sele
cted
from
llam
a de
rive
d im
mun
e lib
rari
es b
y ph
age
disp
lay.
Neu
tral
izin
g V
HH
reco
gniz
ing
diffe
rent
epi
tope
s in
the
rece
ptor
bin
ding
site
s on
the
spik
es w
ith a
ffini
-tie
s in
the
low
nan
omol
ar ra
nge
wer
e id
entifi
ed fo
r all
the
thre
e vi
ruse
s by
vira
l neu
tral
izat
ion
assa
ys. B
y fu
sion
of V
HH
with
var
i-ab
le li
nker
leng
ths,
mul
timer
ic c
onst
ruct
s wer
e m
ade
that
impr
oved
neu
tral
izat
ion
pote
ncie
s up
to 4
000-
fold
for R
SV, 1
500-
fold
fo
r Rab
ies v
irus
and
75-
fold
for I
nflue
nza
H5N
1. Th
e po
tenc
ies o
f the
VH
H c
onst
ruct
s wer
e si
mila
r or b
ette
r tha
n be
st p
erfo
rmin
g m
onoc
lona
l ant
ibod
ies.
The
cros
s pro
tect
ion
capa
city
aga
inst
diff
eren
t vir
al st
rain
s was
als
o im
prov
ed fo
r all
thre
e vi
ruse
s, b
oth
by m
ultiv
alen
t (tw
o or
thre
e id
entic
al V
HH
) and
bip
arat
opic
(tw
o di
ffere
nt V
HH
) con
stru
cts.
By
com
bini
ng a
VH
H n
eutr
aliz
ing
RSV
subt
ype
A, b
ut n
ot su
btyp
e B
with
a p
oorl
y ne
utra
lizin
g V
HH
with
hig
h affi
nity
for s
ubty
pe B
, a b
ipar
atop
ic c
onst
ruct
was
m
ade
with
low
nan
omol
ar n
eutr
aliz
ing
pote
ncy
agai
nst b
oth
subt
ypes
. Tri
vale
nt a
nti-
H5N
1 V
HH
neu
tral
ized
bot
h In
fluen
za
H5N
1 cl
ade1
and
2 in
a p
seud
otyp
e as
say
and
was
ver
y po
tent
in n
eutr
aliz
ing
the
NIB
RG-1
4 In
fluen
za H
5N1
stra
in w
ith IC
(50)
of
9 pi
com
olar
. Biv
alen
t and
bip
arat
opic
con
stru
cts a
gain
st R
abie
s vir
us c
ross
neu
tral
ized
bot
h 10
diff
eren
t Gen
otyp
e 1
stra
ins a
nd
Gen
otyp
e 5.
The
resu
lts sh
ow th
at m
ultim
eriz
atio
n of
VH
H fr
agm
ents
targ
etin
g m
ultip
le e
pito
pes o
n a
vira
l tri
mer
ic sp
ike
prot
ein
is a
pow
erfu
l too
l for
ant
i-vi
ral t
hera
py to
ach
ieve
“bes
t-in
-cla
ss” a
nd b
road
er n
eutr
aliz
atio
n ca
paci
ty.
Hul
tber
g et
al.
2011
Bio
sens
ors
Dec
abod
yC
ellu
lose
filt
erS.
aur
eus
Ant
ibod
y en
gine
erin
g ha
s allo
wed
for t
he ra
pid
gene
ratio
n of
bin
ding
age
nts a
gain
st v
irtu
ally
any
ant
igen
of i
nter
est,
pred
omi-
nant
ly fo
r the
rape
utic
app
licat
ions
. Con
side
rabl
y le
ss a
tten
tion
has b
een
give
n to
the
deve
lopm
ent o
f dia
gnos
tic re
agen
ts a
nd b
io-
sens
ors u
sing
eng
inee
red
antib
odie
s. R
ecen
tly, w
e pr
oduc
ed a
nov
el p
enta
vale
nt b
ispe
cific
ant
ibod
y (i.
e., d
ecab
ody)
by
pent
amer
iz-
ing
two
sing
le-d
omai
n an
tibod
ies (
sdA
bs) t
hrou
gh th
e ve
roto
xin
B su
buni
t (V
TB)
and
foun
d bo
th fu
sion
par
tner
s to
be fu
nctio
nal.
Usi
ng a
sim
ilar a
ppro
ach,
we
have
eng
inee
red
a bi
spec
ific
pent
amer
ic fu
sion
pro
tein
con
sist
ing
of fi
ve sd
Abs
and
five
cel
lulo
se-
bind
ing
mod
ules
(CBM
s) li
nked
via
VT
B. T
o fin
d an
opt
imal
des
ign
form
at, w
e co
nstr
ucte
d si
x bi
spec
ific
pent
amer
s con
sist
ing
of
thre
e di
ffere
nt C
BMs,
fuse
d to
the
Stap
hylo
cocc
us a
ureu
s-sp
ecifi
c hu
man
sdA
b H
VH
P428
, in
both
ori
enta
tions
. One
bis
peci
fic
pent
amer
, con
tain
ing
an N
-ter
min
al C
BM9
and
C-t
erm
inal
HV
HP4
28, w
as so
lubl
e, n
on-a
ggre
gatin
g, a
nd d
id n
ot d
egra
de u
pon
stor
age
at 4
deg
rees
C fo
r ove
r six
mon
ths.
This
mol
ecul
e w
as d
ually
func
tiona
l as i
t bou
nd to
cel
lulo
se-b
ased
filte
rs a
s wel
l as S
. au
reus
cel
ls. W
hen
impr
egna
ted
in c
ellu
lose
filte
rs, t
he b
ispe
cific
pen
tam
er re
cogn
ized
S. a
ureu
s cel
ls in
a fl
ow-t
hrou
gh d
etec
tion
assa
y. Th
e ab
ility
of p
enta
mer
ized
CBM
s to
bind
cel
lulo
se m
ay fo
rm th
e ba
sis o
f an
imm
obili
zatio
n pl
atfo
rm fo
r mul
tival
ent d
ispl
ay
of h
igh-
avid
ity b
indi
ng re
agen
ts o
n ce
llulo
sic
filte
rs fo
r sen
sing
of p
atho
gens
, bio
mar
kers
and
env
iron
men
tal p
ollu
tant
s.
Hus
sack
et
al.
2009
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
490
Chi
mer
ic a
ntig
en
rece
ptor
Ant
i-M
UC
1 na
nobo
dyC
ance
r im
mun
othe
rapy
The
cruc
ial r
ole
of T
lym
phoc
ytes
in a
nti-
tum
or im
mun
ity h
as le
d to
the
deve
lopm
ent o
f nov
el st
rate
gies
that
can
targ
et a
nd a
cti-
vate
T c
ells
aga
inst
tum
or c
ells
. Rec
ombi
nant
DN
A te
chno
logy
has
bee
n us
ed to
gen
erat
e no
n-M
HC
-res
tric
ted
chim
eric
ant
igen
re
cept
ors (
CA
Rs).
Her
e, w
e co
nstr
ucte
d a
pane
l of r
ecom
bina
nt C
AR
that
har
bors
the
anti-
MU
C1
nano
body
and
the
sign
alin
g an
d co
-sig
nalin
g m
oiet
ies (
CD
3 ze
ta/C
D28
) with
diff
eren
t spa
cer r
egio
ns d
eriv
ed fr
om h
uman
IgG
3 w
ith o
ne o
r tw
o re
peat
s of t
he
hing
e se
quen
ce o
r the
hin
ge re
gion
of F
c ga
mm
a RI
I. Th
e Ph
iC31
inte
gras
e sy
stem
was
em
ploy
ed to
inve
stig
ate
if th
e re
com
bina
-tio
n effi
cien
cy c
ould
be
recr
uite
d fo
r hig
h an
d st
able
exp
ress
ion
of T
cel
l chi
mer
ic re
cept
or g
enes
. The
effec
t of n
ucle
ar lo
caliz
atio
n si
gnal
(NLS
) and
two
diffe
rent
pro
mot
ers (
CM
V a
nd C
AG
) on
effica
cy o
f Phi
C31
inte
gras
e in
hum
an T
cel
l lin
es w
as e
valu
ated
. The
pres
ence
of i
nteg
rase
in c
ombi
natio
n w
ith N
LS, m
edia
ted
up to
7.6
and
8.5
fold
incr
ease
s in
CA
R ex
pres
sion
in Z
CH
N-a
ttB
and
ZCH
HN
-att
B ca
sset
te in
tegr
ated
T c
ells
, res
pect
ivel
y. O
ur re
sults
show
ed th
at h
ighl
y effi
cien
t and
stab
le tr
ansd
uctio
n of
the
Jurk
at
cell
line
by P
hiC
31 in
tegr
ase
is a
feas
ible
mod
ality
for g
ener
atin
g an
ti-ca
ncer
chi
mer
ic T
cel
ls fo
r use
in c
ance
r im
mun
othe
rapy
.
Iri-S
ofla
et a
l. 20
11
Nan
obod
y-m
icel
le
conj
ugat
eEp
ider
mal
gro
wth
fact
or
rece
ptor
Lyso
zym
eD
rug
targ
etin
g
The
aim
of t
his s
tudy
was
to d
evel
op p
oly(
ethy
lene
gly
col)-
b-po
ly[N
-(2-
hydr
oxyp
ropy
l) m
etha
cryl
amid
e-la
ctat
e] (m
PEG
-b-
p(H
PMA
m-L
ac(n
)) c
ore-
cros
slin
ked
ther
mos
ensi
tive
biod
egra
dabl
e po
lym
eric
mic
elle
s sui
tabl
e fo
r act
ive
tum
or ta
rget
ing,
by
coup
ling
the
anti-
EGFR
(epi
derm
al g
row
th fa
ctor
rece
ptor
) EG
a1 n
anob
ody
to th
eir s
urfa
ce. T
o th
is e
nd. P
EG w
as fu
nctio
naliz
ed
with
N-s
ucci
nim
idyl
3-(
2-py
ridy
ldith
io)-
prop
iona
te (S
PDP)
to y
ield
a P
DP-
PEG
-b-p
(HPM
Am
-Lac
(n))
blo
ck c
opol
ymer
. Mic
elle
s co
mpo
sed
of 8
0% m
PEG
-b-p
(HPM
Am
-Lac
(n))
and
20%
PD
P-PE
G-b
-p(H
PMA
m-L
ac(n
)) w
ere
prep
ared
and
lyso
zym
e (a
s a m
odel
pr
otei
n) w
as m
odifi
ed w
ith N
-suc
cini
mid
yl-S
-ace
tylth
ioac
etat
e, d
epro
tect
ed w
ith h
ydro
xyla
min
e hy
droc
hlor
ide
and
subs
eque
ntly
co
uple
d to
the
mic
ella
r sur
face
. The
mic
ella
r con
juga
tes w
ere
char
acte
rize
d us
ing
SDS-
PAG
E an
d ge
l per
mea
tion
chro
mat
ogra
phy
(GPC
). U
sing
the
know
ledg
e ob
tain
ed w
ith ly
sozy
me
conj
ugat
ion,
the
EGa1
nan
obod
y w
as c
oupl
ed to
mPE
G/P
DP-
PEG
mic
elle
s an
d th
e co
njug
atio
n w
as su
cces
sful
as d
emon
stra
ted
by w
este
rn b
lot a
nd d
ot b
lot a
naly
sis.
Rho
dam
ine
labe
led
EGa1
-mic
elle
s sh
owed
subs
tant
ially
hig
her b
indi
ng a
s wel
l as u
ptak
e by
EG
FR o
ver-
expr
essi
ng c
ance
r cel
ls (A
431
and
UM
-SC
C-1
4C) t
han
unta
r-ge
ted
rhod
arni
ne la
bele
d m
icel
les.
Inte
rest
ingl
y, no
bin
ding
of t
he n
anob
ody
mic
elle
s was
obs
erve
d to
EG
FR n
egat
ive
cells
(3T
3)
as w
ell a
s to1
4C c
ells
in th
e pr
esen
ce o
f an
exce
ss o
f fre
e na
nobo
dy. Th
is d
emon
stra
tes t
hat t
he b
indi
ng o
f the
nan
obod
y m
icel
les i
s in
deed
by
inte
ract
ion
with
the
EGF
rece
ptor
. In
conc
lusi
on, E
Ga1
dec
orat
ed (m
PEG
/PD
P-PE
G)-
b-(p
HPM
Am
-Lac
(n))
pol
ymer
ic
mic
elle
s are
hig
hly
prom
isin
g sy
stem
s for
act
ive
drug
targ
etin
g.
Tale
lli e
t al
. 201
1
Nan
obod
y-go
ld n
ano-
part
icle
con
juga
tes
HER
2Ph
otot
herm
al th
erap
y
Bran
ched
gol
d na
nopa
rtic
les a
re p
oten
tial p
hoto
ther
mal
ther
apy
agen
ts b
ecau
se o
f the
ir la
rge
abso
rptio
n cr
oss s
ectio
n in
the
near
-in
frar
ed w
indo
w. U
pon
lase
r irr
adia
tion
they
pro
duce
eno
ugh
heat
to d
estr
oy tu
mor
cel
ls. I
n th
is w
ork,
bra
nche
d go
ld n
anop
artic
les a
re
blof
unct
iona
lized
with
nan
obod
ies,
the
smal
lest
fully
func
tiona
l ant
igen
-bin
ding
frag
men
ts e
volv
ed fr
om th
e va
riabl
e do
mai
n, th
e V
HH
, of
a c
amel
hea
vy c
hain
-onl
y an
tibod
y. Th
ese
nano
bodi
es b
ind
to th
e H
ER2
antig
en w
hich
is h
ighl
y ex
pres
sed
on b
reas
t and
ova
rian
canc
er
cells
. Flo
w c
ytom
etric
ana
lysis
and
dar
k fie
ld Im
ages
of H
ER2
posit
ive
SKO
V3
cells
incu
bate
d w
ith a
nti-H
ER2
conj
ugat
ed b
ranc
hed
gold
na
nopa
rtic
les s
how
spec
ific
cell
targ
etin
g. L
aser
Irra
diat
ion
stud
ies r
evea
l tha
t HER
2 po
sitiv
e SK
OV
3 ce
lls e
xpos
ed to
the
anti-
HER
2 ta
r-ge
ted
bran
ched
gol
d na
nopa
rtic
les a
re d
estr
oyed
afte
r five
min
utes
of l
aser
trea
tmen
t at 3
8 W
/cm
2 usin
g a
690
nm c
ontin
uous
wav
e la
ser.
Star
ting
from
a n
anop
artic
le o
ptic
al d
ensit
y of
4, c
ell d
eath
is o
bser
ved,
whe
reas
the
cont
rol s
ampl
es, n
anop
artic
les w
ith a
nti-P
SA n
ano-
bodi
es, n
anop
artic
les o
nly,
and
lase
r onl
y, do
not
show
any
cel
l dea
th. Th
ese
resu
lts su
gges
t tha
t thi
s new
type
of b
ioco
njug
ated
bra
nche
d go
ld n
anop
artic
les a
re e
ffect
ive
antig
en-t
arge
ted
phot
othe
rmal
ther
apeu
tic a
gent
s for
can
cer t
reat
men
t.
Van
de
Broe
k et
al
. 201
1
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
491
Nan
obod
y-lip
osom
e co
njug
ate
Epid
erm
al g
row
th fa
ctor
re
cept
orR
ecep
tor
do
wnr
egul
atio
n
The
epid
erm
al g
row
th fa
ctor
rece
ptor
(EG
FR) i
s a re
cogn
ized
targ
et fo
r tum
or th
erap
y an
d m
onoc
lona
l ant
ibod
ies (
mA
bs, e
.g.
cetu
xim
ab) h
ave
been
dev
elop
ed to
inhi
bit r
ecep
tor a
ctiv
atio
n. B
esid
es b
lock
ing
ligan
d (e
.g. E
GF)
bin
ding
to th
e re
cept
or, r
epor
ts
have
show
n th
at m
Abs
pro
mot
e sl
ow re
cept
or in
tern
aliz
atio
n an
d de
grad
atio
n in
lyso
som
es, i
.e. d
ownr
egul
atio
n. Th
e effi
cacy
of
rece
ptor
dow
nreg
ulat
ion
was
rece
ntly
show
n to
dep
end
on th
e si
ze o
f rec
epto
r clu
ster
s for
med
at t
he c
ell s
urfa
ce. I
n th
is st
udy,
a m
ultiv
alen
t pla
tform
is p
rese
nted
, con
sist
ing
of n
anob
odie
s rec
ogni
zing
the
ecto
dom
ain
of E
GFR
(EG
a1) c
oupl
ed to
PEG
-lip
osom
es, a
nd th
e in
vitr
o an
d in
viv
o eff
ects
of t
his s
yste
m o
n EG
FR in
tern
aliz
atio
n an
d do
wnr
egul
atio
n w
ere
inve
stig
ated
. Nan
o-bo
dies
are
the
smal
lest
func
tiona
l ant
igen
-bin
ding
imm
unog
lobu
lin fr
agm
ents
and
the
EGa1
nan
obod
y ha
s bee
n de
scri
bed
as a
n EG
FR-a
ntag
onis
t. EG
a1-li
poso
mes
(EG
a1-L
) ind
uced
a m
ore
than
90%
rem
oval
of E
GFR
from
the
cell
surf
ace,
as a
resu
lt of
rece
p-to
r int
erna
lizat
ion.
Fur
ther
mor
e, th
is m
assi
ve se
ques
trat
ion
of E
GFR
med
iate
d by
EG
a1-L
lead
to re
cept
or d
egra
datio
n, w
hile
no
degr
adat
ion
was
det
ecte
d w
ith th
e m
onov
alen
t nan
obod
y. Th
e do
wnr
egul
ator
y ca
paci
ty h
ere
repo
rted
was
foun
d to
be
inde
pend
-en
t of t
he e
pito
pe o
n EG
FR re
cogn
ized
by
the
graf
ted
nano
body
, and
exc
lusi
ve to
the
nano
body
-lipo
som
es, a
s ant
i-EG
FR si
ngle
ch
ain
vari
able
frag
men
ts (s
cFv)
cou
pled
to li
poso
mes
wer
e un
able
to in
duce
this
effe
ct. I
mpo
rtan
tly, E
Ga1
-L in
duce
d a
sign
ifica
nt
inhi
bitio
n of
tum
or c
ell p
rolif
erat
ion,
in v
itro,
an
effec
t lik
ely
med
iate
d by
the
com
bina
tion
of re
cept
or d
ownr
egul
atio
n an
d re
cep-
tor a
ntag
onis
m. A
lso
in v
ivo,
EG
FR d
ownr
egul
atio
n w
as o
bser
ved
in tu
mor
s of m
ice
intr
aven
ousl
y in
ject
ed w
ith E
Ga1
-L, i
ndic
atin
g th
at th
is m
ultiv
alen
t pla
tform
blo
cks l
igan
d bi
ndin
g to
the
rece
ptor
and
sim
ulta
neou
sly
indu
ces t
he d
ownr
egul
atio
n of
EG
FR.
Oliv
eira
et
al.
2010
Nan
obod
y-β-
lact
amas
e co
njug
ate
Car
cino
embr
yoni
c an
tige
nC
ance
r th
erap
y
We
iden
tified
a n
anob
ody
with
subn
anom
olar
affi
nity
for t
he h
uman
tum
or-a
ssoc
iate
d ca
rcin
oem
bryo
nic
antig
en. Th
is n
anob
ody
was
con
juga
ted
to E
nter
obac
ter c
loac
ae b
eta-
lact
amas
e, a
nd it
s site
-sel
ectiv
e an
tican
cer p
rodr
ug a
ctiv
atio
n ca
paci
ty w
as e
valu
-at
ed. Th
e co
njug
ate
was
read
ily p
urifi
ed in
hig
h yi
elds
with
out a
ggre
gatio
n or
loss
of f
unct
iona
lity
of th
e co
nstit
uent
s. In
vitr
o ex
peri
men
ts sh
owed
that
the
nano
body
-enz
yme
conj
ugat
e eff
ectiv
ely
activ
ated
the
rele
ase
of p
heny
lene
diam
ine
mus
tard
from
the
ceph
alos
pori
n ni
trog
en m
usta
rd p
rodr
ug 7
-(4-
carb
oxyb
utan
amid
o) c
epha
losp
orin
mus
tard
at t
he su
rfac
e of
car
cino
embr
yoni
c an
tigen
-exp
ress
ing
LS17
4T c
ance
r cel
ls. I
n vi
vo st
udie
s dem
onst
rate
d th
at th
e co
njug
ate
had
an e
xcel
lent
bio
dist
ribu
tion
profi
le
and
indu
ced
regr
essi
ons a
nd c
ures
of e
stab
lishe
d tu
mor
xen
ogra
fts.
The
easy
gen
erat
ion
and
man
ufac
turi
ng y
ield
of n
anob
ody-
base
d co
njug
ates
toge
ther
with
thei
r pot
ent a
ntitu
mor
act
ivity
mak
e na
nobo
dies
pro
mis
ing
vehi
cles
for n
ew g
ener
atio
n ca
ncer
th
erap
eutic
s.
Cor
tez-
Reta
-m
ozo
et
al. 2
004
Nan
obod
y-ap
olip
opro
tein
L-
I con
juga
teTr
ypan
osom
iasi
s th
erap
yIm
mun
otox
in
Hig
h sy
stem
ic d
rug
toxi
city
and
incr
easi
ng p
reva
lenc
e of
dru
g re
sist
ance
ham
pers
effi
cien
t tre
atm
ent o
f hum
an A
fric
an tr
ypan
oso-
mia
sis (
HAT
). H
ence
, dev
elop
men
t of n
ew h
ighl
y sp
ecifi
c tr
ypan
ocid
al d
rugs
is n
eces
sary
. Nor
mal
hum
an se
rum
(NH
S) c
onta
ins
apol
ipop
rote
in L
-I (a
poL-
I), w
hich
lyse
s Afr
ican
tryp
anos
omes
exc
ept r
esis
tant
form
s suc
h as
Try
pano
som
a br
ucei
rhod
esie
nse.
T.
b. rh
odes
iens
e ex
pres
ses t
he a
poL-
I neu
tral
izin
g se
rum
resi
stan
ce-a
ssoc
iate
d (S
RA
) pro
tein
, end
owin
g th
is p
aras
ite w
ith th
e ab
ility
to
infe
ct h
uman
s and
cau
se H
AT. A
trun
cate
d ap
oL-I
(Tr-
apoL
-I) h
as b
een
engi
neer
ed b
y de
letin
g its
SR
A-i
nter
actin
g do
mai
n,
whi
ch m
akes
it ly
tic fo
r T. b
. rho
desi
ense
. Her
e, w
e co
njug
ated
Tr-
apoL
-I w
ith a
sing
le-d
omai
n an
tibod
y (n
anob
ody)
that
effi
cien
tly
targ
ets c
onse
rved
cry
ptic
epi
tope
s of t
he v
aria
nt su
rfac
e gl
ycop
rote
in (V
SG) o
f try
pano
som
es to
gen
erat
e a
new
man
mad
e ty
pe o
f im
mun
otox
in w
ith p
oten
tial f
or tr
ypan
osom
iasi
s the
rapy
. Tre
atm
ent w
ith th
is e
ngin
eere
d co
njug
ate
resu
lted
in c
lear
cur
ativ
e an
d al
levi
atin
g eff
ects
on
acut
e an
d ch
roni
c in
fect
ions
of m
ice
with
bot
h N
HS-
resi
stan
t and
NH
S-se
nsiti
ve tr
ypan
osom
es.
Bara
l et
al. 2
006
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
492
Nan
obod
y-gl
ucos
e
oxid
ase
conj
ugat
eSt
rept
ococ
cus
mut
ans
Ant
imic
robi
al a
gent
s
Enzy
mes
such
as l
acto
pero
xida
se a
nd g
luco
se o
xida
se (G
Ox)
are
use
d as
ant
imic
robi
al a
gent
s in
oral
car
e pr
oduc
ts. Th
eir l
ow
spec
ifici
ties a
nd su
bsta
ntiv
enes
s can
be
redu
ced
by c
oval
ent c
oupl
ing
of a
ntim
icro
bial
mol
ecul
es to
ant
ibod
ies.
Var
iabl
e do
mai
ns
(V-H
H) d
eriv
ed fr
om ll
ama
heav
y-ch
ain
antib
odie
s are
par
ticul
arly
suite
d fo
r suc
h an
app
roac
h. Th
e an
tibod
ies a
re c
ompo
sed
sole
ly o
f hea
vy-c
hain
dim
ers;
ther
efor
e, p
rodu
ctio
n of
act
ive
fusi
on p
rote
ins b
y us
ing
mol
ecul
ar b
iolo
gy-b
ased
tech
niqu
es is
less
co
mpl
icat
ed th
an p
rodu
ctio
n by
use
of c
onve
ntio
nal a
ntib
odie
s. In
this
stud
y, a
fusi
on p
rote
in c
onsi
stin
g of
V-H
H a
nd G
Ox
was
co
nstr
ucte
d an
d ex
pres
sed
by S
acch
arom
yces
cer
evis
iae.
A ll
ama
was
imm
uniz
ed w
ith S
trep
toco
ccus
mut
ans s
trai
n H
G98
2. S
ubse
-qu
ently
, B ly
mph
ocyt
es w
ere
isol
ated
and
cD
NA
frag
men
ts e
ncod
ing
the
V-H
H fr
agm
ents
wer
e ob
tain
ed b
y re
vers
e tr
ansc
ript
ion-
PCR
. Aft
er c
onst
ruct
ion
of a
V-H
H li
brar
y in
Esc
heri
chia
col
i and
scre
enin
g of
the
libra
ry a
gain
st m
utan
s gro
up st
rept
ococ
ci a
nd
Stre
ptoc
occu
s san
guin
is st
rain
s, w
e fo
und
two
V-H
H fr
agm
ents
with
hig
h sp
ecifi
citie
s for
S. m
utan
s str
ains
. A G
Ox
gene
was
lin
ked
to th
e tw
o V
-HH
gen
es a
nd c
lone
d in
to S
. cer
evis
iae
yeas
ts. Th
e ye
asts
exp
ress
ed a
nd se
cret
ed th
e re
com
bina
nt p
rote
ins i
nto
the
grow
th m
ediu
m. Th
e te
st o
f bin
ding
of f
usio
n pr
otei
ns to
ora
l bac
teri
a th
roug
h th
eir V
-HH
frag
men
ts sh
owed
that
S. m
utan
s ha
d be
en sp
ecifi
cally
targ
eted
by
GO
x-S1
20, o
ne o
f the
fusi
on p
rote
in c
onst
ruct
s. A
low
con
cent
ratio
n of
the
fusi
on p
rote
in w
as
also
abl
e to
sele
ctiv
ely
kill
S. m
utan
s with
in 2
0 m
in in
the
pres
ence
of l
acto
pero
xida
se a
nd p
otas
sium
iodi
de. Th
ese
findi
ngs d
emon
-st
rate
that
the
fusi
on p
rote
in G
Ox-
V-H
H is
pot
entia
lly v
alua
ble
in th
e se
lect
ive
killi
ng o
f tar
get b
acte
ria
such
as S
. mut
ans.
Szyn
ol e
t al
. 200
4
Bis
peci
fic a
ntib
ody
Dec
abod
yLi
nker
seq
uenc
eVe
roto
xin
B s
ubun
it
Bisp
ecifi
c an
tibod
ies p
rese
nt u
niqu
e op
port
uniti
es in
term
s of n
ew a
pplic
atio
ns fo
r eng
inee
red
antib
odie
s. H
owev
er, d
esig
ning
id
eal b
ispe
cific
ant
ibod
ies r
emai
ns a
cha
lleng
e. H
ere
we
desc
ribe
a n
ovel
bis
peci
fic a
ntib
ody
mod
el in
whi
ch fi
ve si
ngle
dom
ain
antib
odie
s (sd
Abs
) are
fuse
d vi
a a
linke
r seq
uenc
e to
the
N-t
erm
inus
of t
he v
erot
oxin
B (V
TB)
subu
nit,
a pe
ntam
eriz
atio
n do
mai
n,
and
five
sdA
bs a
re fu
sed
via
a lin
ker s
eque
nce
to th
e V
TB
C-t
erm
inus
. Fift
een
such
dec
aval
ent b
ispe
cific
mol
ecul
es, t
erm
ed
deca
bodi
es, w
ere
cons
truc
ted
and
char
acte
rize
d fo
r the
pur
pose
of i
dent
ifyin
g an
opt
imal
dec
abod
y de
sign
. One
of t
he fi
ftee
n m
olec
ules
exi
sted
in a
non
-agg
rega
ted
deca
vale
nt fo
rm. I
n co
njun
ctio
n w
ith th
e is
olat
ion
of sd
Abs
with
the
desi
red
spec
ifici
ties
from
non
-imm
une
phag
e di
spla
y lib
rari
es, t
he d
ecab
ody
stra
tegy
pro
vide
s a m
eans
of g
ener
atin
g hi
gh a
vidi
ty b
ispe
cific
ant
ibod
y re
agen
ts, w
ith g
ood
phys
ical
pro
pert
ies,
rela
tivel
y qu
ickl
y.
Ston
e et
al
. 200
7
Tabl
e 6H
. App
licat
ion
of s
ingl
e-do
mai
n an
tibo
dy fr
agm
ents
in th
erap
y: h
uman
ised
sin
gle-
dom
ain
anti
body
frag
men
ts
VH
HC
DR
3H
uman
sca
ffold
pro
tein
Gra
ftin
gU
biqu
itin
VH
H is
the
bind
ing
dom
ain
of th
e Ig
G h
eavy
cha
in. S
ome
VH
Hs h
ave
an e
xtre
mel
y lo
ng C
DR3
that
con
trib
utes
to a
ntig
en b
indi
ng.
We
stud
ied
the
antig
en b
indi
ng a
bilit
y of
CD
R3 b
y gr
aftin
g a
CD
R3 fr
om a
n an
tigen
-bin
ding
VH
H o
nto
a no
nbin
ding
VH
H.
cAb-
CA
05-(
1RI8
), th
e C
DR3
-gra
fted
VH
H, h
ad a
n an
tigen
-bin
ding
abi
lity.
To fi
nd a
hum
an sc
affol
d pr
otei
n ac
cept
able
for V
HH
C
DR3
gra
ftin
g, w
e fo
cuse
d on
the
cons
erve
d st
ruct
ure
of V
HH
, esp
ecia
lly th
e N
-ter
min
al a
nd C
-ter
min
al a
min
o ac
id re
sidu
es o
f th
e C
DR3
loop
and
the
Cys
resi
due
of C
DR1
. Hum
an o
rigi
n pr
otei
n st
ruct
ures
with
the
sam
e or
ient
atio
n w
ere
sear
ched
in P
DB
and
ubiq
uitin
was
sele
cted
. Ubi
-(1R
I8),
the
CD
R3-g
raft
ed u
biqu
itin,
had
ant
igen
-bin
ding
abi
lity,
thou
gh th
e affi
nity
was
rela
tivel
y lo
w c
ompa
red
to c
Ab-
CA
05-(
1RI8
). Th
e th
erm
odyn
amic
par
amet
ers o
f Ubi
-(1R
I8) b
indi
ng to
HEW
L w
ere
diffe
rent
from
cA
b-C
A05
-(1R
I8).
Hyd
roge
n-de
uter
ium
exc
hang
e ex
peri
men
ts sh
owed
dec
reas
ed st
abili
ty a
roun
d th
e C
DR3
gra
ftin
g re
gion
of
Ubi
-(1R
I8),
whi
ch m
ight
exp
lain
the
decr
ease
d an
tigen
-bin
ding
abi
lity
and
the
diffe
renc
es in
ther
mod
ynam
ic p
rope
rtie
s. W
e co
n-cl
uded
that
the
orie
ntat
ion
of th
e C
DR3
sequ
ence
of U
bi-(
1RI8
) cou
ld n
ot b
e re
cons
truc
ted
corr
ectly
.
Inou
e et
al
. 201
1
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
493
Aah
II s
corp
ion
toxi
nH
uman
ized
nan
obod
yRe
cent
ly, a
pot
ent A
ahII
scor
pion
toxi
n-ne
utra
lizin
g na
nobo
dy w
as id
entifi
ed. H
owev
er, t
his N
bAah
II10
con
tain
s a si
ngle
Cys
in
its fi
rst a
ntig
en-b
indi
ng lo
op, l
eadi
ng to
Nb
dim
eriz
atio
n up
on p
rolo
nged
stor
age.
In th
is w
ork,
we
first
inve
stig
ate
the
effica
cy o
f N
bAah
II10
var
iant
s in
whi
ch th
is C
ys w
as su
bstit
uted
by
Ala
, Ser
or Th
r. Se
cond
, the
NbA
ahII
10 C
ys/S
er m
utan
t dis
play
ing
the
best
func
tiona
l pro
pert
ies i
s sub
sequ
ently
hum
aniz
ed. I
t is d
emon
stra
ted
that
the
max
imal
ly h
uman
ized
ver
sion
of N
bAah
II10
C
ys/S
er m
aint
ains
its h
igh
affini
ty fo
r the
ant
igen
with
out c
once
ding
muc
h on
exp
ress
ion
yiel
d an
d st
abili
ty. M
ore
impo
rtan
tly, i
ts
neut
raliz
ing
capa
city
is p
rese
rved
as a
ll m
ice
surv
ive
inje
ctio
ns o
f sev
en L
D(5
0) a
nd 5
0 of
mic
e su
rviv
ed n
ine
LD(5
0) o
f the
scor
-pi
on to
xin.
Thus
, thi
s hum
aniz
ed N
b is
the
best
can
dida
te to
dev
elop
a th
erap
y in
hum
an a
gain
st th
e m
ost t
oxic
ven
om c
ompo
und
of o
ne o
f the
mos
t dan
gero
us sc
orpi
ons.
Ben
Abd
er-
raze
k et
al
. 201
1
Hum
aniz
ed n
anob
odie
sLo
op-g
raft
ing
In v
ivo
imag
ing
Scaff
old
Car
cino
embr
yoni
c an
tige
n
Nan
obod
ies a
re a
nov
el ty
pe o
f im
mun
oglo
bulin
like,
ant
igen
-bin
ding
pro
tein
with
ben
efici
al p
harm
acol
ogic
and
pha
rmac
okin
etic
pr
oper
ties t
hat a
re id
eally
suite
d to
targ
etin
g ce
llula
r ant
igen
s for
mol
ecul
ar im
agin
g or
ther
apeu
tic p
urpo
ses.
How
ever
, bec
ause
of
thei
r cam
elid
, non
hum
an o
rigi
n, th
e po
ssib
le im
mun
ogen
icity
of N
anob
odie
s whe
n us
ed in
the
clin
ic is
a c
once
rn. H
ere
we
pres
ent
a ne
w st
rate
gy to
qui
ckly
gen
erat
e hu
man
ized
Nan
obod
ies f
or m
olec
ular
imag
ing
purp
oses
. Met
hods
: We
gene
tical
ly g
raft
ed th
e an
tigen
-bin
ding
loop
s of N
bCEA
5, a
Nan
obod
y w
ith sp
ecifi
city
for t
he c
olon
car
cino
ma
mar
ker c
arci
noem
bryo
nic
antig
en (C
EA),
onto
the
fram
ewor
k of
a h
uman
ized
Nan
obod
y sc
affol
d. Th
is sc
affol
d ha
s bee
n pr
evio
usly
cha
ract
eriz
ed in
our
labo
rato
ry a
s a
stab
le N
anob
ody
that
can
serv
e as
a u
nive
rsal
loop
acc
epto
r for
ant
igen
-bin
ding
loop
s fro
m d
onor
Nan
obod
ies a
nd h
as b
een
addi
-tio
nally
mut
ated
at a
bout
10
cruc
ial s
urfa
ce-e
xpos
ed si
tes t
o re
sem
ble
the
sequ
ence
of h
uman
var
iabl
e im
mun
oglo
bulin
dom
ains
. Th
e 3
reco
mbi
nant
Nan
obod
ies (
NbC
EA5,
hum
aniz
ed sc
affol
d, a
nd h
uman
ized
CEA
5 gr
aft)
wer
e pr
oduc
ed in
bac
teri
a an
d pu
ri-
fied.
Unl
abel
ed a
nd (9
9m)T
c-la
bele
d N
anob
odie
s wer
e bi
oche
mic
ally
cha
ract
eriz
ed in
vitr
o an
d te
sted
as p
robe
s for
SPE
CT
/CT
of
xen
ogra
fted
tum
ors.
Res
ults
: The
succ
ess o
f loo
p-gr
aftin
g w
as c
onfir
med
by
com
pari
ng th
ese
Nan
obod
ies f
or th
eir c
apac
ity to
re
cogn
ize
solu
ble
CEA
pro
tein
in e
nzym
e-lin
ked
imm
unos
orbe
nt a
ssay
and
by
surf
ace
plas
mon
reso
nanc
e an
d to
bin
d to
CEA
-po
sitiv
e LS
174T
col
on c
arci
nom
a ce
lls a
nd C
EA-t
rans
fect
ed b
ut n
ot u
ntra
nsfe
cted
Chi
nese
ham
ster
ova
ry c
ells
in fl
ow c
ytom
etry
. Sp
ecifi
city
of b
indi
ng w
as c
onfir
med
by
com
petit
ion
stud
ies.
All
Nan
obod
ies w
ere
heat
-sta
ble,
cou
ld b
e effi
cien
tly la
bele
d w
ith
(99m
)Tc,
and
reco
gniz
ed b
oth
solu
ble
and
mem
bran
e-bo
und
CEA
pro
tein
in b
indi
ng st
udie
s. F
inal
ly, b
iodi
stri
butio
n ex
peri
men
ts
wer
e pe
rfor
med
with
intr
aven
ousl
y in
ject
ed (9
9m)T
c-la
bele
d N
anob
odie
s in
LS17
4T tu
mor
-bea
ring
mic
e us
ing
pinh
ole
SPEC
T/
mic
ro-C
T. Th
ese
in v
ivo
expe
rim
ents
reve
aled
spec
ifici
ty o
f tum
or ta
rget
ing
and
rapi
d re
nal c
lear
ance
for a
ll N
anob
odie
s, w
ith lo
w
sign
als i
n al
l org
ans b
esid
es th
e ki
dney
s. C
oncl
usio
n: Th
is st
udy
show
s the
pot
ency
of a
ntig
en-b
indi
ng lo
op-g
raft
ing
to e
ffici
ently
ge
nera
te h
uman
ized
Nan
obod
ies t
hat r
etai
n th
eir t
arge
ting
capa
citie
s for
non
inva
sive
in v
ivo
imag
ing
of tu
mor
s.
Vane
y-ck
en e
t al
. 201
0
Uni
vers
al h
uman
ized
na
nobo
dy s
caffo
ldN
anob
odie
s, si
ngle
-dom
ain
antig
en-b
indi
ng fr
agm
ents
of c
amel
id-s
peci
fic h
eavy
-cha
in o
nly
antib
odie
s offe
r spe
cial
adv
anta
ges
in th
erap
y ov
er c
lass
ic a
ntib
ody
frag
men
ts b
ecau
se o
f the
ir sm
alle
r siz
e, ro
bust
ness
, and
pre
fere
nce
to ta
rget
uni
que
epito
pes.
A
Nan
obod
y di
ffers
from
a h
uman
hea
vy c
hain
var
iabl
e do
mai
n in
abo
ut te
n am
ino
acid
s spr
ead
all o
ver i
ts su
rfac
e, fo
ur h
allm
ark
Nan
obod
y-sp
ecifi
c am
ino
acid
s in
the
fram
ewor
k-2
regi
on (p
ositi
ons 4
2, 4
9, 5
0, a
nd 5
2), a
nd a
long
er th
ird
antig
en-b
indi
ng lo
op
(H3)
fold
ing
over
this
are
a. F
or th
erap
eutic
app
licat
ions
the
cam
elid
-spe
cific
am
ino
acid
sequ
ence
s in
the
fram
ewor
k ha
ve to
be
mut
ated
to th
eir h
uman
hea
vy c
hain
var
iabl
e do
mai
n eq
uiva
lent
, i.e
. hum
aniz
ed. W
e pe
rfor
med
this
hum
aniz
atio
n ex
erci
se w
ith
Nan
obod
ies o
f the
subf
amily
that
repr
esen
ts c
lose
to 8
0% o
f all
drom
edar
y-de
rive
d N
anob
odie
s and
inve
stig
ated
the
effec
ts o
n an
tigen
affi
nity
, sol
ubili
ty, e
xpre
ssio
n yi
eld,
and
stab
ility
. It i
s dem
onst
rate
d th
at th
e hu
man
izat
ion
of N
anob
ody-
spec
ific
resi
dues
ou
tsid
e fr
amew
ork-
2 ar
e ne
utra
l to
the
Nan
obod
y pr
oper
ties.
Sur
pris
ingl
y, th
e G
lu-4
9 ->
Gly
and
Arg
-50
-> L
eu h
uman
izat
ion
of
hallm
ark
amin
o ac
ids g
ener
ates
a si
ngle
dom
ain
that
is m
ore
stab
le th
ough
pro
babl
y le
ss so
lubl
e. Th
e ot
her f
ram
ewor
k-2
subs
titu-
tions
, Phe
-42
-> V
al a
nd G
ly/A
la-5
2 ->
Trp
, are
det
rim
enta
l for
ant
igen
affi
nity
, due
to a
repo
sitio
ning
of t
he H
3 lo
op a
s sho
wn
by
thei
r cry
stal
stru
ctur
es. Th
ese
insi
ghts
wer
e us
ed to
iden
tify
a so
lubl
e, st
able
, wel
l exp
ress
ed u
nive
rsal
hum
aniz
ed N
anob
ody
scaf
-fo
ld th
at a
llow
s gra
fts o
f ant
igen
-bin
ding
loop
s fro
m o
ther
Nan
obod
ies w
ith tr
ansf
er o
f the
ant
igen
spec
ifici
ty a
nd a
ffini
ty.
Vin
cke
et a
l. 20
09
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
494
Tabl
e 7.
App
licat
ion
of s
ingl
e-do
mai
n an
tibo
dy fr
agm
ents
in d
iagn
osti
c an
d im
mun
oana
lyti
c m
etho
ds
Non
inva
sive
det
ecti
onTe
chne
tium
-99m
Ath
eros
cler
otic
pla
ques
Sing
le-p
hoto
n em
issi
on
com
pute
d to
mog
raph
y
We
aim
ed to
gen
erat
e, ra
diol
abel
, and
eva
luat
e an
ti-V
CA
M1
nano
bodi
es fo
r non
inva
sive
det
ectio
n of
ath
eros
cler
otic
lesi
ons.
Met
hods
and
Res
ults
: Ten
ant
i-VC
AM
1 na
nobo
dies
wer
e ge
nera
ted,
radi
olab
eled
with
tech
netiu
m-9
9m, a
nd sc
reen
ed in
vitr
o on
mou
se a
nd h
uman
reco
mbi
nant
VC
AM
1 pr
otei
ns a
nd e
ndot
helia
l cel
ls a
nd in
viv
o in
apo
lipop
rote
in E
-defi
cien
t (A
poE(
–/–)
) m
ice.
Ano
ntar
getin
g co
ntro
l nan
obod
y w
as u
sed
in a
ll ex
peri
men
ts to
dem
onst
rate
spec
ifici
ty. A
ll na
nobo
dies
dis
play
ed n
anom
olar
affi
nitie
s for
mur
ine
VC
AM
1. F
low
cyt
omet
ry a
naly
ses u
sing
hum
an h
uman
um
bilic
al v
ein
endo
thel
ial c
ells
indi
cate
d m
urin
e an
d hu
man
VC
AM
1 cr
oss-
reac
tivity
for 6
of 1
0 na
nobo
dies
. The
lead
com
poun
d cA
bVC
AM
1-5
was
cro
ss-r
eact
ive
for h
uman
VC
AM
1 an
d ex
hibi
ted
high
lesi
on-t
o-co
ntro
l (4.
95 ±
0.8
5), l
esio
n-to
-hea
rt (8
.30
± 1.
11),
and
lesi
on-t
o-bl
ood
ratio
s (4.
32 ±
0.4
8) (P
< 0
.05
vers
us c
ontr
ol C
57Bl
/6J m
ice)
. Aor
tic a
rch
athe
rosc
lero
tic le
sion
s of A
poE(
–/–)
mic
e w
ere
succ
essf
ully
iden
tified
by
sing
le-p
hoto
n em
issi
on c
ompu
ted
tom
ogra
phy
imag
ing.
Tc-
99m
-cA
bVC
AM
1-5
bind
ing
spec
ifici
ty w
as d
emon
stra
ted
by in
viv
o co
mpe
titio
n ex
peri
men
ts. A
utor
adio
grap
hy a
nd im
mun
ohis
toch
emis
try
furt
her c
onfir
med
cA
bVC
AM
1-5
upta
ke in
VC
AM
1-po
sitiv
e le
sion
s. C
oncl
usio
ns: Th
e Tc
-99m
-labe
led,
ant
i-VC
AM
1 na
nobo
dy c
AbV
CA
M1-
5 al
low
ed n
onin
vasi
ve d
etec
tion
of V
CA
M1
expr
essi
on
and
disp
laye
d m
ouse
and
hum
an c
ross
-rea
ctiv
ity. Th
eref
ore,
this
stud
y de
mon
stra
tes t
he p
oten
tial o
f nan
obod
ies a
s a n
ew c
lass
of
radi
otra
cers
for c
ardi
ovas
cula
r app
licat
ions
.
Broi
sat e
t al
. 201
2
Tum
our
upta
keTu
mou
r bu
rden
Epid
erm
al g
row
th fa
ctor
re
cept
orB
iolu
min
esce
nce
im
agin
g
We
stud
ied
the
rela
tions
hip
betw
een
tum
or u
ptak
e of
the
epid
erm
al g
row
th fa
ctor
rece
ptor
(EG
FR)-
spec
ific
nano
body
(99m
)Tc
-7C
12 a
nd tu
mor
bur
den
and
eval
uate
d th
e po
ssib
ility
of u
sing
this
pro
be to
mon
itor t
umor
resp
onse
to e
rlot
inib
. The
spec
ific-
ity a
nd a
ffini
ty o
f (99
m)T
c-7C
12 w
as d
eter
min
ed o
n A
431
cells
. Cel
ls e
xpre
ssin
g fir
efly
luci
fera
se w
ere
used
to e
valu
ate
tum
or
burd
en u
sing
bio
lum
ines
cenc
e im
agin
g. W
e ev
alua
ted
the
effec
t of e
rlot
inib
on
tum
or b
urde
n an
d (9
9m)T
c-7C
12 u
ptak
e in
vitr
o as
wel
l as i
n vi
vo. I
n vi
vo b
iolu
min
esce
nce
imag
ing
was
per
form
ed fo
llow
ed b
y pi
nhol
e si
ngle
-pho
ton
emis
sion
com
pute
d to
mog
-ra
phy/
mic
ro-c
ompu
ted
tom
ogra
phy.
(99m
)Tc-
7C12
bin
ds sp
ecifi
cally
to th
e re
cept
or w
ith h
igh
affini
ty (3
.67
± 0.
59 n
M).
Erlo
tinib
re
duce
d tu
mor
upt
ake
and
cell
viab
ility
in a
con
cent
ratio
n-de
pend
ent m
anne
r. Tu
mor
upt
ake
of (9
9m)T
c-7C
12 sh
owed
goo
d co
r-re
latio
n w
ith tu
mor
bur
den.
Erl
otin
ib tr
eatm
ent r
esul
ted
in a
pro
gres
sive
redu
ctio
n of
tum
or b
urde
n an
d tu
mor
upt
ake
of (9
9m)
Tc-7
C12
. (99
m)T
c-7C
12 b
inds
to E
GFR
with
hig
h affi
nity
and
spec
ifici
ty. T
umor
upt
ake
is c
orre
late
d w
ith tu
mor
bur
den.
Qua
ntifi
-ca
tion
of (9
9m)T
c-7C
12 u
ptak
e is
pro
mis
ing
for m
onito
ring
ther
apy
resp
onse
of E
GFR
-exp
ress
ing
tum
ors.
Gai
nkam
et
al.
2011
Nan
opro
beQ
uant
um d
ots
Flow
cyt
omet
ryC
arci
noem
bryo
nic
anti
gen
Bio
psy
sam
ples
Com
mon
stra
tegy
for d
iagn
ostic
s with
qua
ntum
dot
s (Q
Ds)
util
izes
the
spec
ifici
ty o
f mon
oclo
nal a
ntib
odie
s (m
Abs
) for
targ
etin
g.
How
ever
QD
-mA
bs c
onju
gate
s are
not
alw
ays w
ell-
suite
d fo
r thi
s pur
pose
bec
ause
of t
heir
larg
e si
ze. H
ere,
we
engi
neer
ed u
ltr-
asm
all n
anop
robe
s thr
ough
ori
ente
d co
njug
atio
n of
QD
s with
13-
kDa
sing
le-d
omai
n an
tibod
ies (
sdA
bs) d
eriv
ed fr
om ll
ama
IgG
. M
onom
eric
sdA
bs a
re 1
2 tim
es sm
alle
r tha
n m
Abs
and
dem
onst
rate
exc
elle
nt c
apac
ity fo
r ref
oldi
ng. s
dAbs
wer
e ta
gged
with
QD
s th
roug
h an
add
ition
al c
yste
ine
resi
due
inte
grat
ed w
ithin
the
C te
rmin
al o
f the
sdA
b. Th
is a
ppro
ach
allo
wed
us t
o de
velo
p sd
Abs
-Q
D n
anop
robe
s com
pris
ing
four
cop
ies o
f sdA
bs c
oupl
ed w
ith a
QD
in a
hig
hly
orie
nted
man
ner.
sdA
bs-Q
D c
onju
gate
s spe
cific
to
carc
inoe
mbr
yoni
c an
tigen
(CEA
) dem
onst
rate
d ex
celle
nt sp
ecifi
city
of fl
ow c
ytom
etry
qua
ntita
tive
disc
rim
inat
ion
of C
EA-p
ositi
ve
and
CEA
-neg
ativ
e tu
mor
cel
ls. M
oreo
ver,
the
imm
unoh
isto
chem
ical
labe
ling
of b
iops
y sa
mpl
es w
as fo
und
to b
e co
mpa
rabl
e or
ev
en su
peri
or to
the
qual
ity o
btai
ned
with
gol
d st
anda
rd p
roto
cols
of a
nato
mop
atho
logy
pra
ctic
e. sd
Abs
-QD
-ori
ente
d co
njug
ates
as
dev
elop
ed re
pres
ent a
new
gen
erat
ion
of u
ltras
mal
l dia
gnos
tic p
robe
s for
app
licat
ions
in h
igh-
thro
ughp
ut d
iagn
ostic
pla
tform
s.
Sukh
-an
ova
et
al. 2
012
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
495
Chr
omob
ody
Post
tran
slat
iona
l mod
i-fic
atio
ns
The
unde
rsta
ndin
g of
cel
lula
r pro
cess
es a
nd th
eir p
atho
phys
iolo
gica
l alte
ratio
ns re
quir
es c
ompr
ehen
sive
dat
a on
the
abun
danc
e,
dist
ribu
tion,
mod
ifica
tion,
and
inte
ract
ion
of a
ll ce
llula
r com
pone
nts.
On
the
one
hand
, art
ifici
ally
intr
oduc
ed fl
uore
scen
t fus
ion
prot
eins
pro
vide
info
rmat
ion
abou
t the
ir d
istr
ibut
ion
and
dyna
mic
s in
livin
g ce
lls b
ut n
ot a
bout
end
ogen
ous f
acto
rs. O
n th
e ot
her
hand
, ant
ibod
ies c
an d
etec
t end
ogen
ous p
rote
ins,
pos
ttra
nsla
tiona
l mod
ifica
tions
, and
oth
er c
ellu
lar c
ompo
nent
s but
mos
tly in
fix
ed a
nd p
erm
eabi
lized
cel
ls. H
ere
we
high
light
a n
ew te
chno
logy
bas
ed o
n th
e an
tigen
-bin
ding
dom
ain
of h
eavy
-cha
in a
ntib
od-
ies (
VH
H) f
rom
Cam
elid
ae. Th
ese
extr
emel
y st
able
VH
H d
omai
ns c
an b
e pr
oduc
ed in
bac
teri
a, c
oupl
ed to
mat
rice
s, a
nd u
sed
for
affini
ty p
urifi
catio
n an
d pr
oteo
me
stud
ies.
Alte
rnat
ivel
y, th
ese
VH
H d
omai
ns c
an b
e fu
sed
with
fluo
resc
ent p
rote
ins a
nd e
xpre
ssed
in
livi
ng c
ells
. Thes
e flu
ores
cent
ant
igen
-bin
ding
pro
tein
s cal
led
“chr
omob
odie
s” c
an b
e us
ed to
det
ect a
nd tr
ace
prot
eins
and
oth
er
cellu
lar c
ompo
nent
s in
vivo
. Chr
omob
odie
s can
, in
prin
cipl
e, d
etec
t any
ant
igen
ic st
ruct
ure,
incl
udin
g po
sttr
ansl
atio
nal m
odifi
-ca
tions
, and
ther
eby
dram
atic
ally
exp
and
the
qual
ity a
nd q
uant
ity o
f inf
orm
atio
n th
at c
an b
e ga
ther
ed in
hig
h-co
nten
t ana
lysi
s. D
epen
ding
on
the
epito
pe c
hose
n, c
hrom
obod
ies c
an a
lso
be u
sed
to m
odul
ate
prot
ein
func
tion
in li
ving
cel
ls.
Schm
idt-
ha
ls et
al.
2010
deG
radF
PG
reen
fluo
resc
ent p
ro-
tein
Prot
ein
knoc
kout
The
use
of g
enet
ic m
utat
ions
to st
udy
prot
ein
func
tions
in v
ivo
is a
cen
tral
par
adig
m o
f mod
ern
biol
ogy.
Rece
nt a
dvan
ces i
n re
vers
e ge
netic
s suc
h as
RN
A in
terf
eren
ce a
nd m
orph
olin
os a
re w
idel
y us
ed to
furt
her a
pply
this
par
adig
m. N
ever
thel
ess,
such
syst
ems a
ct
upst
ream
of t
he p
rote
ic le
vel,
and
prot
ein
depl
etio
n de
pend
s on
the
turn
over
rate
of t
he e
xist
ing
targ
et p
rote
ins.
Her
e w
e pr
esen
t de
Gra
dFP,
a g
enet
ical
ly e
ncod
ed m
etho
d fo
r dir
ect a
nd fa
st d
eple
tion
of ta
rget
gre
en fl
uore
scen
t pro
tein
(GFP
) fus
ions
in a
ny
euka
ryot
ic g
enet
ic sy
stem
. This
met
hod
is u
nive
rsal
bec
ause
it re
lies o
n an
evo
lutio
nari
ly h
ighl
y co
nser
ved
euka
ryot
ic fu
nctio
n, th
e ub
iqui
tin p
athw
ay. I
t is t
race
able
, bec
ause
the
GFP
tag
can
be u
sed
to m
onito
r the
pro
tein
kno
ckou
t. In
man
y ca
ses,
it is
a re
ady-
to-
use
solu
tion,
as G
FP p
rote
in-t
rap
stoc
k co
llect
ions
are
bei
ng g
ener
ated
in D
roso
phila
mel
anog
aste
r and
in D
anio
reri
o.
Cau
ssi-
nus e
t al.
2012
Alz
heim
er’s
dise
ase
Pept
ide
conf
orm
atio
nA
myl
oid
beta
Neu
roto
xici
ty
Neu
roto
xic
olig
omer
s of a
myl
oid
beta
(A b
eta)
pep
tide
have
bee
n in
crim
inat
ed in
the
path
ogen
esis
of A
lzhe
imer
’s di
seas
e. F
urth
er
expl
orat
ion
of th
is is
sue
has b
een
ham
pere
d to
this
dat
e by
the
fact
that
all
prev
ious
ly d
escr
ibed
ant
i-A
bet
a an
tibod
ies a
re u
nabl
e to
dis
crim
inat
e be
twee
n th
e di
ffere
nt c
onfo
rmat
ions
of t
he p
eptid
e (o
ligom
ers,
pro
tofib
rils
and
fibr
ils).
Her
e w
e de
scri
be th
e ge
n-er
atio
n of
nov
el c
amel
id si
ngle
-cha
in b
indi
ng d
omai
ns (V
HH
s) th
at re
cogn
izes
spec
ifica
lly lo
w m
olec
ular
-wei
ght (
MW
) olig
omer
s. Th
ree
VH
H sp
ecifi
c fo
r A b
eta
wer
e ob
tain
ed fr
om a
n im
mun
ized
alp
aca
phag
e di
spla
y lib
rary
. Tw
o w
ere
able
to re
cogn
ize
sele
c-tiv
ely
intr
aneu
rona
l A b
eta
olig
omer
s: fu
rthe
rmor
e, o
ne o
f the
m, V
31-1
, pre
vent
ed A
bet
a-in
duce
d ne
urot
oxic
ity a
nd in
hibi
ted
fibri
l for
mat
ion.
This
stud
y co
nfirm
s tha
t VH
Hs m
ay re
cogn
ize
non-
conv
entio
nal e
pito
pes a
nd il
lust
rate
s the
ir p
oten
tial f
or th
e im
mun
odia
gnos
tic o
f dis
ease
s due
to p
rote
in a
ccum
ulat
ion.
Lafa
ye e
t al
. 200
9
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
496
Hum
aniz
ed n
anob
odie
sLo
op-g
raft
ing
In v
ivo
imag
ing
Scaff
old
Car
cino
embr
yoni
c an
tige
n
Nan
obod
ies a
re a
nov
el ty
pe o
f im
mun
oglo
bulin
like,
ant
igen
-bin
ding
pro
tein
with
ben
efici
al p
harm
acol
ogic
and
pha
rmac
okin
etic
pr
oper
ties t
hat a
re id
eally
suite
d to
targ
etin
g ce
llula
r ant
igen
s for
mol
ecul
ar im
agin
g or
ther
apeu
tic p
urpo
ses.
How
ever
, bec
ause
of
thei
r cam
elid
, non
hum
an o
rigi
n, th
e po
ssib
le im
mun
ogen
icity
of N
anob
odie
s whe
n us
ed in
the
clin
ic is
a c
once
rn. H
ere
we
pres
ent
a ne
w st
rate
gy to
qui
ckly
gen
erat
e hu
man
ized
Nan
obod
ies f
or m
olec
ular
imag
ing
purp
oses
. Met
hods
: We
gene
tical
ly g
raft
ed th
e an
tigen
-bin
ding
loop
s of N
bCEA
5, a
Nan
obod
y w
ith sp
ecifi
city
for t
he c
olon
car
cino
ma
mar
ker c
arci
noem
bryo
nic
antig
en (C
EA),
onto
the
fram
ewor
k of
a h
uman
ized
Nan
obod
y sc
affol
d. Th
is sc
affol
d ha
s bee
n pr
evio
usly
cha
ract
eriz
ed in
our
labo
rato
ry a
s a
stab
le N
anob
ody
that
can
serv
e as
a u
nive
rsal
loop
acc
epto
r for
ant
igen
-bin
ding
loop
s fro
m d
onor
Nan
obod
ies a
nd h
as b
een
addi
-tio
nally
mut
ated
at a
bout
10
cruc
ial s
urfa
ce-e
xpos
ed si
tes t
o re
sem
ble
the
sequ
ence
of h
uman
var
iabl
e im
mun
oglo
bulin
dom
ains
. Th
e 3
reco
mbi
nant
Nan
obod
ies (
NbC
EA5,
hum
aniz
ed sc
affol
d, a
nd h
uman
ized
CEA
5 gr
aft)
wer
e pr
oduc
ed in
bac
teri
a an
d pu
ri-
fied.
Unl
abel
ed a
nd (9
9m)T
c-la
bele
d N
anob
odie
s wer
e bi
oche
mic
ally
cha
ract
eriz
ed in
vitr
o an
d te
sted
as p
robe
s for
SPE
CT
/CT
of
xen
ogra
fted
tum
ors.
Res
ults
: The
succ
ess o
f loo
p-gr
aftin
g w
as c
onfir
med
by
com
pari
ng th
ese
Nan
obod
ies f
or th
eir c
apac
ity to
re
cogn
ize
solu
ble
CEA
pro
tein
in e
nzym
e-lin
ked
imm
unos
orbe
nt a
ssay
and
by
surf
ace
plas
mon
reso
nanc
e an
d to
bin
d to
CEA
-po
sitiv
e LS
174T
col
on c
arci
nom
a ce
lls a
nd C
EA-t
rans
fect
ed b
ut n
ot u
ntra
nsfe
cted
Chi
nese
ham
ster
ova
ry c
ells
in fl
ow c
ytom
etry
. Sp
ecifi
city
of b
indi
ng w
as c
onfir
med
by
com
petit
ion
stud
ies.
All
Nan
obod
ies w
ere
heat
-sta
ble,
cou
ld b
e effi
cien
tly la
bele
d w
ith
(99m
)Tc,
and
reco
gniz
ed b
oth
solu
ble
and
mem
bran
e-bo
und
CEA
pro
tein
in b
indi
ng st
udie
s. F
inal
ly, b
iodi
stri
butio
n ex
peri
men
ts
wer
e pe
rfor
med
with
intr
aven
ousl
y in
ject
ed (9
9m)T
c-la
bele
d N
anob
odie
s in
LS17
4T tu
mor
-bea
ring
mic
e us
ing
pinh
ole
SPEC
T/
mic
ro-C
T. Th
ese
in v
ivo
expe
rim
ents
reve
aled
spec
ifici
ty o
f tum
or ta
rget
ing
and
rapi
d re
nal c
lear
ance
for a
ll N
anob
odie
s, w
ith lo
w
sign
als i
n al
l org
ans b
esid
es th
e ki
dney
s. C
oncl
usio
n: Th
is st
udy
show
s the
pot
ency
of a
ntig
en-b
indi
ng lo
op-g
raft
ing
to e
ffici
ently
ge
nera
te h
uman
ized
Nan
obod
ies t
hat r
etai
n th
eir t
arge
ting
capa
citie
s for
non
inva
sive
in v
ivo
imag
ing
of tu
mor
s.
Vane
y-ck
en e
t al
. 201
0
HER
-2B
reas
t can
cer
Non
inva
sive
imag
ing
Acc
urat
e de
term
inat
ion
of tu
mor
hum
an e
pide
rmal
gro
wth
fact
or re
cept
or 2
(HER
2)-s
tatu
s in
brea
st c
ance
r pat
ient
s is p
ossi
ble
via
noni
nvas
ive
imag
ing,
pro
vide
d ad
equa
te tr
acer
s are
use
d. In
this
stud
y, w
e de
scri
be th
e ge
nera
tion
of a
pan
el o
f 38
nano
bodi
es,
smal
l HER
2-bi
ndin
g fr
agm
ents
that
are
der
ived
from
hea
vy-c
hain
-onl
y an
tibod
ies r
aise
d in
an
imm
uniz
ed d
rom
edar
y. In
sear
ch
of a
lead
com
poun
d, a
subs
et o
f nan
obod
ies w
as b
ioch
emic
ally
cha
ract
eriz
ed in
dep
th a
nd p
recl
inic
ally
test
ed fo
r use
as t
race
rs
for i
mag
ing
of x
enog
raft
ed tu
mor
s. Th
e se
lect
ed c
ompo
und,
2Rs
15d,
was
foun
d to
be
stab
le a
nd to
inte
ract
spec
ifica
lly w
ith H
ER2
reco
mbi
nant
pro
tein
and
HER
2-ex
pres
sing
cel
ls in
ELI
SA, s
urfa
ce p
lasm
on re
sona
nce,
flow
cyt
omet
ry, a
nd ra
diol
igan
d bi
ndin
g st
udie
s with
low
nan
omol
ar a
ffini
ties,
and
did
not
com
pete
with
ant
i-HER
2 th
erap
eutic
ant
ibod
ies t
rast
uzum
ab a
nd p
ertu
zum
ab.
Sing
le-p
hoto
n-em
issi
on c
ompu
ted
tom
ogra
phy
(SPE
CT
) im
agin
g qu
antifi
catio
n an
d bi
odis
trib
utio
n an
alys
es sh
owed
that
(99m
)Tc
-labe
led
2Rs1
5d h
as a
hig
h tu
mor
upt
ake
in 2
HER
2+ tum
or m
odel
s, fa
st b
lood
cle
aran
ce, l
ow a
ccum
ulat
ion
in n
onta
rget
org
ans
exce
pt k
idne
ys, a
nd h
igh
conc
omita
nt tu
mor
-to-
bloo
d an
d tu
mor
-to-
mus
cle
ratio
s at 1
h a
fter
intr
aven
ous i
njec
tion.
Thes
e va
lues
w
ere
dram
atic
ally
low
er fo
r an
irre
leva
nt c
ontr
ol (9
9m)T
c-na
nobo
dy a
nd fo
r (99
m)T
c-2R
s15d
targ
etin
g a
HER
2– tum
or.
Vane
y-ck
en e
t al
. 201
1b
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
497
VH
H-b
ased
imm
unoa
ssay
3-ph
enox
yben
zoic
aci
dP
yret
hroi
d in
sect
icid
es
We
expr
esse
d V
HH
s fro
m a
n im
mun
ized
alp
aca
and
deve
lope
d a
VH
H-b
ased
imm
unoa
ssay
usi
ng 3
-phe
noxy
benz
oic
acid
(3-P
BA),
a m
ajor
met
abol
ite o
f pyr
ethr
oid
inse
ctic
ides
as a
mod
el sy
stem
. A p
hage
VH
H li
brar
y w
as c
onst
ruct
ed, a
nd se
ven
VH
H c
lone
s w
ere
sele
cted
by
com
petit
ive
bind
ing
with
3-P
BA. Th
e be
st im
mun
oass
ay d
evel
oped
with
one
of t
hese
VH
Hs s
how
ed a
n IC
50 o
f 1.
4 ng
/ml (
limit
of d
etec
tion
(LO
D) =
0.1
ng/
ml).
Thes
e pa
ram
eter
s wer
e fu
rthe
r im
prov
ed b
y us
ing
the
phag
e bo
rne
VH
H, I
C50
=
0.1
ng/m
l and
LO
D =
0.0
1 ng
/ml.
Both
ass
ays s
how
ed a
sim
ilar t
oler
ance
to m
etha
nol a
nd d
imet
hyls
ulfo
xide
up
to 5
0% in
ass
ay
buffe
r. Th
e as
say
was
hig
hly
spec
ific
to 3
-PBA
and
its 4
-hyd
roxy
late
d de
riva
tive,
4-h
ydro
xy 3
-PBA
, (15
0% c
ross
reac
tivity
) with
neg
-lig
ible
cro
ss re
activ
ity w
ith o
ther
test
ed st
ruct
ural
ana
logu
es, a
nd th
e re
cove
ry fr
om sp
iked
uri
ne sa
mpl
e ra
nged
from
80
to 1
12%
. In
con
clus
ion,
a h
ighl
y sp
ecifi
c an
d se
nsiti
ve V
HH
for 3
-PBA
was
dev
elop
ed u
sing
sequ
ence
s fro
m im
mun
ized
alp
aca
and
phag
e di
spla
y te
chno
logy
for a
ntib
ody
sele
ctio
n.
Kim
et a
l. 20
12
Bot
ulin
um n
euro
toxi
nTo
xin
neut
raliz
ing
ca
pabi
lity
Hig
h te
mpe
ratu
res
Back
grou
nd: Th
ere
are
curr
ently
7 k
now
n se
roty
pes o
f bot
ulin
um n
euro
toxi
n (B
oNT
) cla
ssifi
ed u
pon
non-
cros
s rea
ctiv
ity o
f ne
utra
lizin
g im
mun
oglo
bulin
s. N
on-n
eutr
aliz
ing
imm
unog
lobu
lins,
how
ever
, can
exh
ibit
cros
s-re
activ
ities
bet
wee
n 2
or m
ore
sero
type
s, p
artic
ular
ly m
osai
c fo
rms,
whi
ch c
an h
ampe
r the
dev
elop
men
t of h
ighl
y sp
ecifi
c im
mun
oass
ays,
esp
ecia
lly if
bas
ed o
n po
lycl
onal
ant
iser
a. H
ere
we
empl
oy fa
cile
reco
mbi
nant
ant
ibod
y te
chno
logy
to su
btra
ctiv
ely
sele
ct li
gand
s to
each
of t
he 7
BoN
T
sero
type
s, re
sulti
ng in
pop
ulat
ions
with
ver
y hi
gh sp
ecifi
city
for t
heir
inte
nded
sero
type
. Met
hods
and
Fin
ding
s: A
sing
le ll
ama
was
imm
uniz
ed w
ith a
coc
ktai
l of 7
BoN
T to
xoid
s to
gene
rate
a p
hage
dis
play
libr
ary
of si
ngle
dom
ain
antib
odie
s (sd
Ab,
VH
H o
r na
nobo
dies
) whi
ch w
ere
sele
cted
on
live
toxi
ns. R
esul
ting
sdA
b w
ere
capa
ble
of d
etec
ting
both
toxi
n an
d to
xin
com
plex
with
the
best
com
bina
tions
abl
e to
det
ect 1
00s-
10s o
f pg
per 5
0 µl
sam
ple
in a
liqu
id b
ead
arra
y. Th
e m
ost s
ensi
tive
sdA
b w
ere
com
bine
d in
a
hept
aple
x as
say
to id
entif
y ea
ch o
f the
BoN
T se
roty
pes i
n bu
ffer a
nd m
ilk a
nd to
a le
sser
ext
ent i
n ca
rrot
juic
e, o
rang
e ju
ice
and
cola
. Sev
eral
ant
i-A(1
) sdA
b re
cogn
ized
A2
com
plex
, sho
win
g th
at su
btyp
e cr
oss-
reac
tivity
with
in a
sero
type
was
evi
dent
. Man
y of
our
sdA
b co
uld
act a
s bot
h ca
ptor
and
trac
er fo
r sev
eral
toxi
n an
d to
xin
com
plex
es su
gges
ting
sdA
b ca
n be
use
d as
arc
hite
c-tu
ral p
robe
s to
indi
cate
BoN
T o
ligom
eris
atio
n. S
ix o
f 14
anti-
A c
lone
s exh
ibite
d in
hibi
tion
of S
NA
P-25
cle
avag
e in
the
neur
o-2A
as
say
indi
catin
g so
me
sdA
b ha
d to
xin
neut
raliz
ing
capa
bilit
ies.
Man
y sd
Ab
wer
e al
so sh
own
to b
e re
fold
able
aft
er e
xpos
ure
to h
igh
tem
pera
ture
s in
cont
rast
to p
olyc
lona
l ant
iser
a, a
s mon
itore
d by
cir
cula
r dic
hroi
sm. C
oncl
usio
ns: O
ur p
anel
of m
olec
ular
ly fl
exib
le
antib
odie
s sho
uld
not o
nly
serv
e as
a g
ood
star
ting
poin
t for
rugg
ediz
ing
assa
ys a
nd in
hibi
tors
, but
ena
ble
the
intr
icat
e ar
chite
c-tu
res o
f BoN
T to
xins
and
com
plex
es to
be
prob
ed m
ore
exte
nsiv
ely.
Con
way
et
al.
2010
ELIS
ATo
xin
SdA
b-al
kalin
e
phos
phat
ase
fusi
on
Nai
ve li
brar
ies o
f sin
gle
dom
ain
antib
odie
s (sd
Abs
) ena
ble
rapi
d is
olat
ion
of b
inde
rs to
nea
rly
any
targ
et. Th
ese
bind
ers,
how
ever
, la
ck th
e be
nefit
s bes
tow
ed b
y in
viv
o affi
nity
mat
urat
ion
and
typi
cally
hav
e lo
w a
ffini
ty to
war
d th
eir t
arge
ts. W
e ex
pres
sed
five
low
-affi
nity
toxi
n bi
ndin
g sd
Abs
, pre
viou
sly
sele
cted
from
a n
aive
libr
ary
deri
ved
from
var
iabl
e re
gion
s of l
lam
a he
avy
chai
n-on
ly
antib
odie
s, a
s fus
ions
with
a h
yper
activ
e m
utan
t Esc
heri
chia
col
i alk
alin
e ph
osph
atas
e (A
P) a
nd e
xam
ined
the
impa
ct o
n ap
pare
nt
affini
ty a
nd u
tility
. AP
spon
tane
ousl
y di
mer
izes
in so
lutio
n, e
ffect
ivel
y di
mer
izin
g th
e fu
sed
sdA
bs, i
mpa
rtin
g av
idity
in p
lace
of t
he
low
er a
ffini
ty m
onom
eric
inte
ract
ions
. The
sdA
b-A
P fu
sion
als
o co
mbi
nes t
he ta
rget
reco
gniti
on d
omai
n w
ith a
sign
al tr
ansd
uctio
n do
mai
n, c
omm
only
use
d in
enz
yme-
linke
d im
mun
osor
bent
ass
ays (
ELIS
As)
. The
func
tiona
l affi
nity
of t
he sd
Ab-
AP
fusi
ons,
oft
en
incr
ease
d by
a fa
ctor
of 1
0 ov
er u
nfus
ed sd
Abs
, and
thei
r util
ity a
s tra
cer r
eage
nts i
n EL
ISA
s was
dra
mat
ical
ly im
prov
ed, g
ivin
g lim
its o
f det
ectio
n of
300
ng/
ml o
r les
s, w
here
as p
aren
tal s
dAbs
gav
e no
dis
cern
ible
sign
al a
t the
toxi
n co
ncen
trat
ions
test
ed. Th
e fu
sion
of s
dAbs
to A
P pr
esen
ts a
val
uabl
e ro
ute
to fa
cilit
ate
the
impl
emen
tatio
n of
sdA
b-ba
sed
imm
unor
eage
nts r
apid
ly se
lect
ed
from
exi
stin
g na
ive
libra
ries
tow
ard
new
or e
mer
ging
thre
ats.
Swai
n et
al
. 201
1
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
498
ELIS
AR
icin
Bot
ulin
um A
toxi
nC
y-3
fluor
esce
nt d
ye
Prev
ious
ly, w
e se
lect
ed sd
Ab
that
wer
e sp
ecifi
c fo
r bot
h ri
cin
and
botu
linum
A (B
oNT
A) t
oxin
com
plex
from
pha
ge d
ispl
ay li
brar
-ie
s of s
dAb
and
eval
uate
d th
e so
lubl
y ex
pres
sed
prot
ein.
Her
e, p
hage
-dis
play
ed sd
Ab
wer
e us
ed a
s rep
orte
r rea
gent
s and
com
pare
d to
solu
ble,
unf
used
sdA
b. W
e fo
und
that
usi
ng p
hage
-dis
play
ed sd
Ab
as re
port
er e
lem
ents
in im
mun
oass
ay fo
rmat
s gav
e im
prov
ed
dete
ctio
n ov
er u
sing
unf
used
, sol
uble
sdA
b re
port
ers.
In e
nzym
e-lin
ked
imm
unos
orbe
nt a
ssay
s (EL
ISA
s), t
he lo
wes
t lev
el o
f tox
in
dete
cted
for b
oth
rici
n an
d Bo
NT
A to
xoid
com
plex
was
dec
reas
ed b
y on
e to
two
orde
rs o
f mag
nitu
de u
sing
pha
ge-d
ispl
ayed
sdA
b as
repo
rter
reag
ents
. Use
of t
he p
hage
pre
serv
ed th
e ab
ility
to d
iscr
imin
ate
rici
n an
d RC
A12
0 by
at l
east
a fa
ctor
of 1
0 fo
ld. I
n an
eff
ort t
o re
duce
the
num
ber o
f ste
ps in
the
assa
ys, w
e di
rect
ly la
bele
d ph
age
disp
layi
ng sd
Ab
with
a C
y-3
fluor
esce
nt d
ye. S
igna
l w
as g
reat
ly d
ecre
ased
usi
ng th
e dy
e-la
bele
d ph
age
com
pare
d to
bio
tinyl
ated
pha
ge fo
llow
ed b
y st
rept
avid
in-p
hyco
eryt
hrin
. In
thes
e as
says
the
use
of p
hage
-dis
play
ed sd
Ab
give
s mor
e se
nsiti
ve d
etec
tion
than
solu
ble
sdA
b al
one,
how
ever
dir
ectly
dye
labe
ling
the
phag
e fa
iled
to p
rovi
de re
spon
ses o
f a si
mila
r mag
nitu
de.
Gol
dman
et
al.
2010
Ant
ibod
y m
icro
arra
ysSu
rfac
e ex
pres
sion
Fibr
obla
st g
row
th fa
ctor
re
cept
or 1
The
prep
arat
ion
of e
ffect
ive
conv
entio
nal a
ntib
ody
mic
roar
rays
dep
ends
on
the
avai
labi
lity
of h
igh
qual
ity m
ater
ial a
nd o
n th
e co
rrec
t acc
essi
bilit
y of
the
antib
ody
activ
e m
oiet
ies f
ollo
win
g th
eir i
mm
obili
zatio
n on
the
supp
ort s
lide.
We
show
that
spot
ting
bact
eria
that
exp
ose
reco
mbi
nant
ant
ibod
ies o
n th
eir e
xter
nal s
urfa
ce d
irec
tly o
n na
nost
ruct
ured
-TiO
(2) o
r epo
xy sl
ides
(pur
i-fic
atio
n-in
depe
nden
t mic
roar
ray
– PI
M) i
s a si
mpl
e an
d re
liabl
e al
tern
ativ
e fo
r pre
pari
ng se
nsiti
ve a
nd sp
ecifi
c m
icro
arra
ys fo
r an
tigen
det
ectio
n. V
aria
ble
dom
ains
of s
ingl
e he
avy-
chai
n an
tibod
ies (
VH
Hs)
aga
inst
fibr
obla
st g
row
th fa
ctor
rece
ptor
1 (F
GFR
1)
wer
e us
ed to
cap
ture
the
antig
en d
ilute
d in
seru
m o
r BSA
solu
tion.
The
FGFR
1 de
tect
ion
was
per
form
ed b
y ei
ther
dir
ect a
ntig
en
labe
ling
or u
sing
a sa
ndw
ich
syst
em in
whi
ch F
GFR
1 w
as fi
rst b
ound
to it
s ant
ibod
y an
d su
cces
sive
ly id
entifi
ed u
sing
a la
bele
d FG
F.
In b
oth
case
s the
sign
al d
istr
ibut
ion
with
in e
ach
spot
was
uni
form
and
spot
mor
phol
ogy
regu
lar.
The
sign
al-t
o-no
ise
ratio
of t
he
sign
al w
as e
xtre
mel
y el
evat
ed a
nd th
e sp
ecifi
city
of t
he sy
stem
was
pro
ved
stat
istic
ally
. The
LOD
of t
he sy
stem
for t
he a
ntig
en w
as
calc
ulat
ed b
eing
0.4
ng/
ml a
nd th
e dy
nam
ic ra
nge
betw
een
0.4
ng/m
l and
10
μg/m
l. Th
e m
icro
arra
ys p
repa
red
with
bac
teri
a ex
pos-
ing
antib
odie
s rem
ain
fully
func
tiona
l for
at l
east
31
clay
s aft
er sp
ottin
g. W
e fin
ally
dem
onst
rate
d th
at th
e m
etho
d is
suita
ble
for
othe
r ant
igen
-ant
ibod
y pa
irs a
nd e
xpec
t tha
t it c
ould
be
easi
ly a
dapt
ed to
furt
her a
pplic
atio
ns su
ch a
s the
dis
play
of s
cFv
and
IgG
an
tibod
ies o
r the
aut
oant
ibod
y de
tect
ion
usin
g pr
otei
n.
De
Mar
ni e
t al
. 201
2
Com
peti
tive
ass
ayTr
iclo
carb
anU
sing
tric
loca
rban
(TC
C) a
s a m
odel
hap
ten,
we
foun
d th
at c
onve
ntio
nal a
ntib
odie
s, Ig
G1
frac
tion,
reac
ted
with
free
TC
C w
ith a
hi
gher
rela
tive
affini
ty (I
C(5
0) 5
1.0
ng/m
l) th
an d
id th
e sd
Abs
(IgG
2 an
d Ig
G3,
497
and
370
ng/
ml,
resp
ectiv
ely)
. A V
HH
libr
ary
was
pre
pare
d, a
nd b
y el
utio
n of
pha
ge w
ith li
miti
ng c
once
ntra
tions
of T
CC
and
com
petit
ive
sele
ctio
n of
bin
ders
, we
wer
e ab
le to
is
olat
e hi
gh-a
ffini
ty c
lone
s, K
(D) 0
.98–
1.37
nM
(SPR
), w
hich
allo
wed
dev
elop
men
t of a
com
petit
ive
assa
y fo
r TC
C w
ith a
n IC
(50)
=
3.5
ng/m
l (11
nM
). Th
is re
pres
ents
a 1
00-f
old
impr
ovem
ent w
ith re
gard
to th
e pe
rfor
man
ce o
f the
sdA
b se
rum
frac
tion,
and
it
is 1
00-f
old
bett
er th
an th
e IC
(50)
att
aine
d w
ith o
ther
ant
ihap
ten
VH
Hs r
epor
ted
thus
far.
Des
pite
the
mod
est o
vera
ll an
tihap
ten
sdA
bs re
spon
se in
llam
as, a
smal
l sub
popu
latio
n of
hig
h-affi
nity
VH
Hs i
s gen
erat
ed th
at c
an b
e is
olat
ed b
y ca
refu
l des
ign
of th
e se
lect
ion
proc
ess.
Taba
res-
da R
osa
et a
l. 20
11
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
499
Den
atur
ing
agen
tsC
elia
c pa
tien
tsPr
olam
inG
liadi
nEL
ISA
Food
inte
nded
for c
elia
c pa
tient
s’ co
nsum
ptio
n m
ust b
e an
alyz
ed fo
r the
pre
senc
e of
toxi
c pr
olam
ins u
sing
hig
h de
lect
abili
ty te
sts.
Thou
gh 6
0% e
than
ol is
the
mos
t com
mon
ly u
sed
solv
ent f
or p
rola
min
s ext
ract
ion,
2-m
erca
ptoe
than
ol (2
-ME)
and
gua
nidi
nium
chl
o-ri
de (G
uHC
l) ca
n be
add
ed to
incr
ease
pro
tein
reco
very
. How
ever
, eth
anol
and
den
atur
ing
agen
ts in
terf
ere
with
ant
igen
reco
gniti
on
whe
n co
nven
tiona
l ant
ibod
ies a
re u
sed.
In th
e pr
esen
t wor
k, a
new
met
hod
for g
liadi
ns q
uant
ifica
tion
is sh
own.
The
met
hod
is b
ased
on
the
sele
ctio
n of
llam
a si
ngle
dom
ain
antib
ody
frag
men
ts a
ble
to o
pera
te u
nder
den
atur
ing
cond
ition
s. Si
x ou
t of 2
8 V
HH
-pha
ges
obta
ined
reta
ined
thei
r bin
ding
cap
acity
in 1
5% e
than
ol. S
elec
ted
clon
es p
rese
nted
a lo
ng C
DR3
regi
on c
onta
inin
g tw
o ad
ditio
nal
cyst
eine
s tha
t cou
ld b
e re
spon
sibl
e fo
r the
hig
her s
tabi
lity.
One
of t
he c
lone
s (na
med
VH
H26
) was
fully
ope
rativ
e in
the
pres
ence
of
15%
eth
anol
, 0.5
% 2
-ME,
and
0.5
M G
uHC
l. C
aptu
re E
LISA
usi
ng V
HH
26 w
as a
ble
to d
etec
t glia
dins
in sa
mpl
es sh
own
as n
egat
ives
by
con
vent
iona
l ELI
SA. Th
eref
ore,
this
new
stra
tegy
app
ears
as a
n ex
celle
nt p
latfo
rm fo
r qua
ntita
tive
dete
rmin
atio
n of
pro
tein
s or a
ny
othe
r im
mun
ogen
ic c
ompo
und,
in th
e pr
esen
ce o
f den
atur
ing
agen
ts, w
hen
spec
ific
reco
gniti
on u
nits
with
hig
h st
abili
ty a
re re
quire
d.
Don
a et
al
. 201
0
ELIS
ATa
enia
sol
ium
Cys
tice
rcos
is
Taen
ia so
lium
cys
ticer
cosi
s is a
maj
or h
elm
inth
zoo
nosi
s in
deve
lopi
ng c
ount
ries
. Pig
s are
the
inte
rmed
iate
hos
ts m
edia
ting
tran
s-m
issi
on o
f inf
ectio
n. S
peci
fic a
ssay
s to
diag
nose
livi
ng c
ysts
in p
igs a
re la
ckin
g. Th
e m
onod
onal
-bas
ed a
ntig
en d
etec
tion
ELIS
A is
ge
nus-
spec
ific
and
cros
s-re
actio
ns w
ith T
aeni
a hy
datig
ena
ham
per t
he u
se o
f thi
s tes
t to
scre
en p
igs.
We,
ther
efor
e, a
imed
to in
tro-
duce
nan
obod
ies,
cam
elid
-der
ived
sing
le-d
omai
n an
tibod
ies s
peci
fic fo
r T. s
oliu
m c
ystic
erco
sis,
to d
evel
op u
nam
bigu
ous t
ests
. N
anob
odie
s wer
e cl
oned
follo
win
g im
mun
izat
ion
of tw
o dr
omed
arie
s with
T. s
oliu
m a
ntig
en a
nd e
ight
T. s
oliu
m-s
peci
fic n
ano-
bodi
es w
ere
sele
cted
aft
er p
hage
dis
play
. Thei
r bin
ding
cha
ract
eris
tics a
nd p
oten
tial f
or th
e di
agno
sis o
f por
cine
cys
ticer
cosi
s wer
e in
vest
igat
ed. Th
e na
nobo
dies
do
not c
ross
-rea
ct w
ith T
hyd
atig
ena,
Tae
nia
sagi
nata
, Tae
nia
cras
sice
ps o
r Tri
chin
ella
spir
alis
and
w
ere
cate
gori
zed
into
four
epi
tope
-bin
ding
gro
ups.
The
targ
et p
rote
in w
as id
entifi
ed a
s 14
kDa
diag
nost
ic g
lyco
prot
ein
(Ts1
4), b
ut
the
nano
bodi
es a
lso
reac
ted
with
oth
er p
rote
ins o
f the
sam
e fa
mily
. Nan
obod
ies w
ere
test
ed in
a sa
ndw
ich
ELIS
A w
ith c
yst fl
uid,
an
d on
e pa
rtic
ular
nan
obod
y de
tect
ed it
s cog
nate
seru
m a
ntig
ens i
n a
spec
ies-
spec
ific
inhi
bitio
n EL
ISA
. Con
side
ring
thei
r ben
efi-
cial
pro
duct
ion
and
stab
ility
pro
pert
ies,
thes
e hi
ghly
spec
ific
nano
bodi
es c
onst
itute
a p
rom
isin
g to
ol to
dia
gnos
e cy
stic
erco
sis a
fter
fu
rthe
r im
prov
emen
t of t
he se
nsiti
vity
and
futu
re a
ssay
val
idat
ion.
Dec
k-er
s et a
l. 20
09
Bio
sens
orEb
olav
irus
Clin
ical
and
en
viro
nmen
tal
sam
ples
IgN
AR
Hae
mor
rhag
ic fe
ver
Mem
bers
of t
he g
enus
Ebo
lavi
rus c
ause
fulm
inat
ing
outb
reak
s of d
isea
se in
hum
an a
nd n
on-h
uman
pri
mat
e po
pula
tions
with
a
mor
talit
y ra
te u
p to
90%
. To
faci
litat
e ra
pid
dete
ctio
n of
thes
e pa
thog
ens i
n cl
inic
al a
nd e
nvir
onm
enta
l sam
ples
, rob
ust r
eage
nts
capa
ble
of p
rovi
ding
sens
itive
and
spec
ific
dete
ctio
n ar
e re
quir
ed. I
n th
is w
ork
reco
mbi
nant
ant
ibod
y lib
rari
es w
ere
gene
rate
d fr
om
mur
ine
(sin
gle
chai
n va
riab
le d
omai
n fr
agm
ent;
scFv
) and
nur
se sh
ark,
Gin
glym
osto
ma
cirr
atum
(IgN
AR
V) h
osts
imm
unis
ed
with
Zai
re e
bola
viru
s. Th
is p
rovi
des t
he fi
rst r
ecor
ded
IgN
AR
V re
spon
se a
gain
st a
par
ticul
ate
antig
en in
the
nurs
e sh
ark.
Bot
h m
urin
e sc
Fv a
nd sh
ark
IgN
AR
V li
brar
ies w
ere
pann
ed b
y ph
age
disp
lay
tech
nolo
gy to
iden
tify
usef
ul a
ntib
odie
s for
the
gene
ratio
n of
imm
unol
ogic
al d
etec
tion
reag
ents
. Tw
o m
urin
e sc
Fv w
ere
show
n to
hav
e sp
ecifi
city
to th
e Z
aire
ebo
lavi
rus v
iral
mat
rix
prot
ein
VP4
0. T
wo
isol
ated
IgN
AR
V w
ere
show
n to
bin
d to
the
vira
l nuc
leop
rote
in (N
P) a
nd to
cap
ture
via
ble
Zai
re e
bola
viru
s with
a h
igh
degr
ee o
f sen
sitiv
ity. A
ssay
s dev
elop
ed w
ith Ig
NA
R V
cro
ss-r
eact
ed to
Res
ton
ebol
avir
us, S
udan
ebo
lavi
rus a
nd B
undi
bugy
o eb
ola-
viru
s. D
espi
te th
is b
road
reac
tivity
, nei
ther
of I
gNA
R V
show
ed re
activ
ity to
Cot
e di
voir
e eb
olav
irus
. IgN
AR
V w
as su
bsta
ntia
lly
mor
e re
sist
ant t
o ir
reve
rsib
le th
erm
al d
enat
urat
ion
than
mur
ine
scFv
and
mon
oclo
nal I
gG in
a c
ompa
rativ
e te
st. Th
e de
mon
stra
ble
robu
stne
ss o
f the
IgN
AR
V d
omai
ns m
ay o
ffer e
nhan
ced
utili
ty a
s im
mun
olog
ical
det
ectio
n re
agen
ts in
fiel
dabl
e bi
osen
sor a
pplic
a-tio
ns fo
r use
in tr
opic
al o
r sub
trop
ical
cou
ntri
es w
here
out
brea
ks o
f Ebo
lavi
rus h
aem
orrh
agic
feve
r occ
ur.
Goo
d-ch
ild e
t al
. 201
1
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
500
Bio
sens
orD
ecab
ody
Cel
lulo
se fi
lter
sS.
aur
eus
Ant
ibod
y en
gine
erin
g ha
s allo
wed
for t
he ra
pid
gene
ratio
n of
bin
ding
age
nts a
gain
st v
irtu
ally
any
ant
igen
of i
nter
est,
pred
omi-
nant
ly fo
r the
rape
utic
app
licat
ions
. Con
side
rabl
y le
ss a
tten
tion
has b
een
give
n to
the
deve
lopm
ent o
f dia
gnos
tic re
agen
ts a
nd b
io-
sens
ors u
sing
eng
inee
red
antib
odie
s. R
ecen
tly, w
e pr
oduc
ed a
nov
el p
enta
vale
nt b
ispe
cific
ant
ibod
y (i.
e., d
ecab
ody)
by
pent
amer
iz-
ing
two
sing
le-d
omai
n an
tibod
ies (
sdA
bs) t
hrou
gh th
e ve
roto
xin
B su
buni
t (V
TB)
and
foun
d bo
th fu
sion
par
tner
s to
be fu
nctio
nal.
Usi
ng a
sim
ilar a
ppro
ach,
we
have
eng
inee
red
a bi
spec
ific
pent
amer
ic fu
sion
pro
tein
con
sist
ing
of fi
ve sd
Abs
and
five
cel
lulo
se-
bind
ing
mod
ules
(CBM
s) li
nked
via
VT
B. T
o fin
d an
opt
imal
des
ign
form
at, w
e co
nstr
ucte
d si
x bi
spec
ific
pent
amer
s con
sist
ing
of
thre
e di
ffere
nt C
BMs,
fuse
d to
the
Stap
hylo
cocc
us a
ureu
s-sp
ecifi
c hu
man
sdA
b H
VH
P428
, in
both
ori
enta
tions
. One
bis
peci
fic
pent
amer
, con
tain
ing
an N
-ter
min
al C
BM9
and
C-t
erm
inal
HV
HP4
28, w
as so
lubl
e, n
on-a
ggre
gatin
g, a
nd d
id n
ot d
egra
de u
pon
stor
age
at fo
ur d
egre
es C
for o
ver s
ix m
onth
s. Th
is m
olec
ule
was
dua
lly fu
nctio
nal a
s it b
ound
to c
ellu
lose
-bas
ed fi
lters
as w
ell a
s S.
aure
us c
ells
. Whe
n im
preg
nate
d in
cel
lulo
se fi
lters
, the
bis
peci
fic p
enta
mer
reco
gniz
ed S
. aur
eus c
ells
in a
flow
-thr
ough
det
ectio
n as
say.
The
abili
ty o
f pen
tam
eriz
ed C
BMs t
o bi
nd c
ellu
lose
may
form
the
basi
s of a
n im
mob
iliza
tion
plat
form
for m
ultiv
alen
t dis
play
of
hig
h-av
idity
bin
ding
reag
ents
on
cellu
losi
c fil
ters
for s
ensi
ng o
f pat
hoge
ns, b
iom
arke
rs a
nd e
nvir
onm
enta
l pol
luta
nts.
Hus
sack
et
al.
2009
SRP-
sens
orEL
ISA
M. t
uber
culo
sis
Phag
e di
spla
y se
lect
ion
stra
tegi
esH
eat s
hock
pro
tein
Back
grou
nd: R
ecom
bina
nt a
ntib
odie
s are
pow
erfu
l too
ls in
eng
inee
ring
of n
ovel
dia
gnos
tics.
Due
to th
e sm
all s
ize
and
stab
le
natu
re o
f lla
ma
antib
ody
dom
ains
sele
cted
ant
ibod
ies c
an se
rve
as a
det
ectio
n re
agen
t in
mul
tiple
xed
and
sens
itive
ass
ays f
or M
. tu
berc
ulos
is. M
etho
dolo
gy/P
rinc
ipal
Fin
ding
s: A
ntib
odie
s for
Myc
obac
teri
um tu
berc
ulos
is (M
. tb)
reco
gniti
on w
ere
rais
ed in
A
lpac
a, a
nd, b
y ph
age
disp
lay,
reco
mbi
nant
var
iabl
e do
mai
ns o
f hea
vy-c
hain
ant
ibod
ies (
VH
H) b
indi
ng to
M. t
uber
culo
sis a
ntig
ens
wer
e is
olat
ed. T
wo
phag
e di
spla
y se
lect
ion
stra
tegi
es w
ere
follo
wed
: one
dir
ect s
elec
tion
usin
g se
mi-
puri
fied
prot
ein
antig
en, a
nd a
de
plet
ion
stra
tegy
with
lysa
tes,
aim
ing
to a
void
cro
ss-r
eact
ion
to o
ther
myc
obac
teri
a. B
oth
pann
ing
met
hods
sele
cted
a se
t of b
ind-
ers w
ith w
idel
y di
fferi
ng c
ompl
emen
tari
ty d
eter
min
ing
regi
ons.
Sel
ecte
d re
com
bina
nt V
HH
s wer
e pr
oduc
ed in
E. c
oli a
nd sh
own
to b
ind
imm
obili
zed
lysa
te in
dir
ect E
nzym
elin
ked
Imm
unos
orbe
nt A
ssay
(ELI
SA) t
ests
and
solu
ble
antig
en b
y su
rfac
e pl
asm
on
reso
nanc
e (S
PR) a
naly
sis.
All
test
ed V
HH
s wer
e sp
ecifi
c fo
r tub
ercu
losi
s-ca
usin
g m
ycob
acte
ria
(M. t
uber
culo
sis,
M. b
ovis
) and
ex
clus
ivel
y re
cogn
ized
an
imm
unod
omin
ant 1
6 kD
a he
at sh
ock
prot
ein
(hsp
). Th
e hi
ghes
t affi
nity
VH
H h
ad a
dis
soci
atio
n co
nsta
nt
(KD
) of 4
× 1
0(–1
0) M
. Con
clus
ions
/Sig
nific
ance
: A b
road
set o
f diff
eren
t lla
ma
antib
odie
s spe
cific
for 1
6 kD
a he
at sh
ock
prot
ein
of M
. tub
ercu
losi
s is a
vaila
ble.
This
pro
tein
is h
ighl
y st
able
and
abu
ndan
t in
M. t
uber
culo
sis.
The
VH
H th
at d
etec
t thi
s pro
tein
are
ap
plie
d in
a ro
bust
SPR
sens
or fo
r ide
ntifi
catio
n of
tube
rcul
osis
-cau
sing
myc
obac
teri
a.
Trill
ing
et a
l. 20
11
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
501
Tabl
e 8.
Oth
er p
rosp
ecti
ve u
ses
of s
ingl
e-do
mai
n an
tibo
dy fr
agm
ents
Cry
stal
str
uctu
reEd
itos
ome
Tryp
anos
oma
bruc
ei
The
para
site
Try
pano
som
a br
ucei
, the
cau
sativ
e ag
ent o
f sle
epin
g si
ckne
ss a
cros
s sub
-Sah
aran
Afr
ica,
dep
ends
on
a re
mar
kabl
e U
-inse
rtio
n/de
letio
n RN
A e
ditin
g pr
oces
s in
its m
itoch
ondr
ion.
A a
ppro
xim
atel
y 20
S m
ulti-
prot
ein
com
plex
, cal
led
the
edito
-so
me,
is a
n es
sent
ial m
achi
nery
for e
ditin
g pr
e-m
RNA
mol
ecul
es e
ncod
ing
the
maj
ority
of m
itoch
ondr
ial p
rote
ins.
Edi
toso
mes
co
ntai
n a
com
mon
cor
e of
twel
ve p
rote
ins w
here
six
OB-
fold
inte
ract
ion
prot
eins
, cal
led
A1-
A6,
pla
y a
cruc
ial r
ole.
Her
e, w
e re
port
th
e st
ruct
ure
of tw
o si
ngle
-str
and
nucl
eic
acid
-bin
ding
OB-
fold
s fro
m in
tera
ctio
n pr
otei
ns A
3 an
d A
6 th
at su
rpri
sing
ly, f
orm
a h
et-
erod
imer
. Cry
stal
gro
wth
requ
ired
the
assi
stan
ce o
f an
anti-
A3
nano
body
as a
cry
stal
lizat
ion
chap
eron
e. U
nexp
ecte
dly,
this
ant
i-A
3 na
nobo
dy b
inds
to b
oth
A3(
OB)
and
A6,
des
pite
onl
y si
mila
r to
40%
am
ino
acid
sequ
ence
iden
tity
betw
een
the
OB-
fold
s of A
3 an
d A
6. Th
e A
3(O
B)-A
6 he
tero
dim
er b
urie
s 35%
mor
e su
rfac
e ar
ea th
an th
e A
6 ho
mod
imer
. This
is a
ttri
bute
d m
ainl
y to
the
pres
ence
of
a co
nser
ved
Pro-
rich
loop
in A
3(O
B). Th
e im
plic
atio
ns o
f the
A3(
OB)
-A6
hete
rodi
mer
, and
of a
dim
er o
f het
erod
imer
s obs
erve
d in
th
e cr
ysta
ls, f
or th
e ar
chite
ctur
e of
the
edito
som
e ar
e pr
ofou
nd, r
esul
ting
in a
pro
posa
l of a
‘five
OB-
fold
cen
ter’
in th
e co
re o
f the
ed
itoso
me.
Park
et
al. 2
012
X-r
ay c
ryst
allo
grap
hyPr
ion
In s
itu
prot
eoly
sis
Mic
rose
ed m
atri
x sc
reen
ing
Prio
n pr
otei
ns (P
rPs)
are
diffi
cult
to c
ryst
alliz
e, p
roba
bly
due
to th
eir i
nher
ent fl
exib
ility
. Sev
eral
PrP
s str
uctu
res h
ave
been
solv
ed
by n
ucle
ar m
agne
tic re
sona
nce
(NM
R) te
chni
ques
; how
ever
, onl
y th
ree
stru
ctur
es w
ere
solv
ed b
y X
-ray
cry
stal
logr
aphy
. Her
e w
e co
mbi
ned
in-s
itu p
rote
olys
is w
ith a
utom
ated
mic
rose
ed m
atri
x sc
reen
ing
(MM
S) to
cry
stal
lize
two
diffe
rent
PrP
(C)-
nano
body
(N
b) c
ompl
exes
. Nan
obod
ies a
re si
ngle
-dom
ain
antib
odie
s der
ived
from
hea
vy-c
hain
-onl
y an
tibod
ies o
f cam
elid
s. In
itial
cry
stal
-liz
atio
n sc
reen
ing
cond
ition
s usi
ng in
situ
pro
teol
ysis
of m
ouse
pri
on (2
3–23
0) in
com
plex
with
a n
anob
ody
(Nb_
PrP_
01) g
ave
thin
ne
edle
agg
rega
tes,
whi
ch w
ere
of p
oor d
iffra
ctio
n qu
ality
. Nex
t, w
e us
ed th
ese
mic
rocr
ysta
ls a
s nuc
lean
ts fo
r aut
omat
ed M
MS.
G
ood-
qual
ity c
ryst
als w
ere
obta
ined
from
mou
se P
rP (8
9–23
0)/N
b_Pr
P_01
, bel
onge
d to
the
mon
oclin
ic sp
ace
grou
p P
1 21
1, w
ith
unit-
cell
para
met
ers a
= 5
9.13
, b =
63.
80, c
= 6
9.79
ang
stro
m, β
= 1
01.9
6° a
nd d
iffra
cted
to 2
.1 a
ngst
rom
reso
lutio
n us
ing
sync
hro-
tron
radi
atio
n. H
uman
PrP
(90–
231)
/Nb_
PrP_
01 c
ryst
als b
elon
ged
to th
e m
onoc
linic
spac
e gr
oup
C2,
with
uni
t-ce
ll pa
ram
eter
s a
= 13
1.86
, b =
45.
78, c
= 4
5.09
ang
stro
m, β
= 9
6.23
° and
diff
ract
ed to
1.5
ang
stro
m re
solu
tion.
This
com
bine
d st
rate
gy b
enefi
ts
from
the
pow
er o
f the
MM
S te
chni
que
with
out s
uffer
ing
from
the
draw
back
s of t
he in
situ
pro
teol
ysis
. It p
rove
d to
be
a su
cces
sful
st
rate
gy to
cry
stal
lize
PrP-
nano
bodi
es c
ompl
exes
and
cou
ld b
e ex
ploi
ted
for t
he c
ryst
alliz
atio
n of
oth
er d
ifficu
lt an
tigen
antib
ody
com
plex
es.
Abs
kha-
ron
et a
l. 20
11
X-r
ay c
ryst
allo
grap
hyC
onfo
rmat
iona
l in
term
edia
tes
Am
yloi
doge
nesi
sB
eta
2-m
icro
glob
ulin
Ato
mic
-leve
l str
uctu
ral i
nves
tigat
ion
of th
e ke
y co
nfor
mat
iona
l int
erm
edia
tes o
f am
yloi
doge
nesi
s rem
ains
a c
halle
nge.
Her
e w
e de
mon
stra
te th
e ut
ility
of n
anob
odie
s to
trap
and
cha
ract
eriz
e in
term
edia
tes o
f bet
a 2-
mic
rogl
obul
in (b
eta
2m) a
myl
oido
gene
sis
by X
-ray
cry
stal
logr
aphy
. For
this
pur
pose
, we
sele
cted
five
sing
le d
omai
n an
tibod
ies t
hat b
lock
the
fibri
lloge
nesi
s of a
pro
teol
ytic
am
yloi
doge
nic
frag
men
t of b
eta
2m (D
elta
N6
beta
2m
). Th
e cr
ysta
l str
uctu
re o
f Del
ta N
6 be
ta 2
m in
com
plex
with
one
of t
hese
na
nobo
dies
(Nb2
4) id
entifi
es d
omai
n sw
appi
ng a
s a p
laus
ible
mec
hani
sm o
f sel
f-as
soci
atio
n of
this
am
yloi
doge
nic
prot
ein.
In
the
swap
ped
dim
er, t
wo
exte
nded
hin
ge lo
ops-
corr
espo
ndin
g to
the
hept
apet
ide
NH
VT
LSQ
that
form
s am
yloi
d in
isol
atio
n-ar
e un
mas
ked
and
fold
into
a n
ew tw
o-st
rand
ed a
ntip
aral
lel b
eta-
shee
t. Th
e be
ta-s
tran
ds o
f thi
s she
et a
re p
rone
to se
lf-as
soci
ate
and
stac
k pe
rpen
dicu
lar t
o th
e di
rect
ion
of th
e st
rand
s to
build
larg
e in
term
olec
ular
bet
a-sh
eets
that
run
para
llel t
o th
e ax
is o
f gro
win
g ol
igom
ers,
pro
vidi
ng a
n el
onga
tion
mec
hani
sm b
y se
lf-te
mpl
ated
gro
wth
.
Dom
an-
ska
et a
l. 20
11
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
502
Tran
spor
t sys
tem
sIm
mun
oglo
bulin
re
cept
orC
arri
er s
yste
mTr
ansc
ytos
isEp
ithe
lial m
onol
ayer
The
poly
mer
ic im
mun
oglo
bulin
rece
ptor
(pIg
R) e
nsur
es th
e tr
ansp
ort o
f dim
eric
imm
unog
lobu
lin A
(dIg
A) a
nd p
enta
mer
ic im
mu-
nogl
obul
in M
(pIg
M) a
cros
s epi
thel
ia to
the
muc
osal
laye
r of f
or e
xam
ple
the
inte
stin
es a
nd th
e lu
ngs v
ia tr
ansc
ytos
is. P
er d
ay th
e hu
man
pIg
R m
edia
tes t
he e
xcre
tion
of 2
to 5
gra
ms o
f dIg
A in
to th
e m
ucos
a of
lum
inal
org
ans.
This
syst
em c
ould
pro
ve u
sefu
l fo
r the
rapi
es a
imin
g at
exc
retio
n of
com
poun
ds in
to th
e m
ucos
a. H
ere
we
inve
stig
ated
the
use
of th
e va
riab
le d
omai
n of
cam
elid
de
rive
d he
avy
chai
n on
ly a
ntib
odie
s, a
lso
know
n as
VH
Hs o
r Nan
obod
ies,
targ
etin
g th
e hu
man
pIg
R, a
s a tr
ansp
ort s
yste
m a
cros
s ep
ithel
ial c
ells
. We
show
that
VH
Hs d
irec
ted
agai
nst t
he h
uman
pIg
R ar
e ab
le to
bin
d th
e re
cept
or w
ith h
igh
affini
ty (s
imila
r to
1 nM
) and
that
they
com
pete
with
the
natu
ral l
igan
d, d
IgA
. In
a tr
ansc
ytos
is a
ssay
bot
h na
tive
and
phag
e-bo
und
VH
H w
ere
only
abl
e to
get
acr
oss p
olar
ized
MD
CK
cel
ls th
at e
xpre
ss th
e hu
man
pIg
R ge
ne in
a b
asol
ater
al to
api
cal f
ashi
on. I
ndic
atin
g th
at th
e V
HH
s ar
e ab
le to
tran
sloc
ate
acro
ss e
pith
elia
and
to ta
ke a
long
larg
e pa
rtic
les o
f car
go. F
urth
erm
ore,
by
mak
ing
mul
tival
ent V
HH
s we
wer
e ab
le to
enh
ance
the
tran
spor
t of t
he c
ompo
unds
bot
h in
a M
DC
K-hp
IgR
and
Cac
o-2
cell
syst
em, p
roba
bly
by in
duci
ng re
cep-
tor c
lust
erin
g. Th
ese
resu
lts sh
ow th
at V
HH
s can
be
used
as a
car
rier
syst
em to
exp
loit
the
hum
an p
IgR
tran
scyt
otic
syst
em a
nd
that
mul
tival
ent c
ompo
unds
are
abl
e to
sign
ifica
ntly
enh
ance
the
tran
spor
t acr
oss e
pith
elia
l mon
olay
ers.
Emm
er-
son
et a
l. 20
11
Tran
spor
t sys
tem
sM
icel
leEG
FRD
oxor
ubic
inEn
caps
ulat
ion
effici
ency
Cor
e-cr
ossl
inke
d th
erm
osen
sitiv
e an
d bi
odeg
rada
ble
poly
mer
ic m
icel
les w
ere
activ
ely
targ
eted
to E
GFR
-ove
rexp
ress
ing
canc
er
cells
by
conj
ugat
ing
an a
nti-E
GFR
nan
obod
y on
the
surf
ace
of th
e m
icel
les.
A m
etha
cryl
ated
dox
orub
icin
der
ivat
ive
cont
aini
ng a
n ac
id se
nsiti
ve h
ydra
zone
spac
er w
as e
ncap
sula
ted
and
subs
eque
ntly
cov
alen
tly a
ttac
hed
duri
ng c
ore-
cros
slin
king
of t
he m
icel
les.
Enca
psul
atio
n effi
cien
cy w
as 6
0% a
nd d
oxor
ubic
in (D
OX
) was
com
plet
ely
rele
ased
aft
er 2
4 h
at p
H 5
, whi
le h
ardl
y an
y D
OX
was
re
leas
ed a
t pH
7.4
. DO
X-lo
aded
mic
elle
s sho
wed
toxi
city
sim
ilar t
o fr
ee d
oxor
ubic
in to
war
ds o
vari
an c
arci
nom
a ce
lls.
Tale
lli e
t al
. 201
0
Dru
g de
liver
y ve
hicl
eSo
dalis
glo
ssin
idiu
sPa
ratr
ansg
enes
isTr
ypan
osom
a br
ucei
Back
grou
nd: S
odal
is g
loss
inid
ius,
a g
ram
-neg
ativ
e ba
cter
ial e
ndos
ymbi
ont o
f the
tset
se fl
y, ha
s bee
n pr
opos
ed a
s a p
oten
tial i
n vi
vo
drug
del
iver
y ve
hicl
e to
con
trol
tryp
anos
ome
para
site
dev
elop
men
t in
the
fly, a
n ap
proa
ch k
now
n as
par
atra
nsge
nesi
s. D
espi
te th
is
inte
rest
of S
. glo
ssin
idiu
s as a
par
atra
nsge
nic
plat
form
org
anis
m in
tset
se fl
ies,
few
pot
entia
l effe
ctor
mol
ecul
es h
ave
been
iden
tified
so
far a
nd to
dat
e no
ne o
f the
se m
olec
ules
hav
e be
en su
cces
sful
ly e
xpre
ssed
in th
is b
acte
rium
. Res
ults
: In
this
stud
y, S.
glo
ssin
idiu
s w
as tr
ansf
orm
ed to
exp
ress
a si
ngle
dom
ain
antib
ody,
(Nan
obod
y) N
b_A
n33,
that
effi
cien
tly ta
rget
s con
serv
ed c
rypt
ic e
pito
pes
of th
e va
rian
t sur
face
gly
copr
otei
n (V
SG) o
f the
par
asite
Try
pano
som
a br
ucei
. Nex
t, w
e an
alyz
ed th
e ca
pabi
lity
of tw
o pr
edic
ted
secr
etio
n si
gnal
s to
dire
ct th
e ex
trac
ellu
lar d
eliv
ery
of si
gnifi
cant
leve
ls o
f act
ive
Nb_
An3
3. W
e sh
ow th
at th
e pe
lB le
ader
pep
tide
was
succ
essf
ul in
dir
ectin
g th
e ex
port
of f
ully
func
tiona
l Nb_
An3
3 to
the
peri
plas
m o
f S. g
loss
inid
ius r
esul
ting
in si
gnifi
cant
leve
ls
of e
xtra
cellu
lar r
elea
se. F
inal
ly, S
. glo
ssin
idiu
s exp
ress
ing
pelB
Nb_
An3
3 ex
hibi
ted
no si
gnifi
cant
redu
ctio
n in
term
s of fi
tnes
s, de
term
ined
by
in v
itro
grow
th k
inet
ics,
com
pare
d to
the
wild
-typ
e st
rain
. Con
clus
ions
: Thes
e da
ta a
re th
e fir
st d
emon
stra
tion
of
the
expr
essi
on a
nd e
xtra
cellu
lar r
elea
se o
f fun
ctio
nal t
rypa
noso
me-
inte
rfer
ing
Nan
obod
ies i
n S.
glo
ssin
idiu
s. F
urth
erm
ore,
Sod
alis
st
rain
s tha
t effi
cien
tly re
leas
ed th
e eff
ecto
r pro
tein
wer
e no
t affe
cted
in th
eir g
row
th, s
ugge
stin
g th
at th
ey m
ay b
e co
mpe
titiv
e w
ith
endo
geno
us m
icro
biot
a in
the
mid
gut e
nvir
onm
ent o
f the
tset
se fl
y. C
olle
ctiv
ely,
thes
e da
ta re
info
rce
the
notio
n fo
r the
pot
entia
l of
S. g
loss
inid
ius t
o be
dev
elop
ed in
to a
par
atra
nsge
nic
plat
form
org
anis
m.
De
Voog
ht
et a
l. 20
12
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
503
Blo
od-b
rain
bar
rier
Dru
g bi
odis
trib
utio
nIn
viv
o im
agin
gLi
poso
me
nano
carr
ier
The
deve
lopm
ent o
f im
agin
g an
d th
erap
eutic
age
nts a
gain
st n
euro
nal t
arge
ts is
ham
pere
d by
the
limite
d ac
cess
of p
robe
s int
o th
e ce
ntra
l ner
vous
syst
em a
cros
s the
blo
od b
rain
bar
rier
(BBB
). Th
e ev
alua
tion
of d
rug
pene
trat
ion
into
the
brai
n in
exp
erim
enta
l m
odel
s oft
en re
quir
es c
ompl
ex p
roce
dure
s, in
clud
ing
drug
radi
olab
elin
g, a
s wel
l as d
eter
min
atio
ns in
mul
tiple
ani
mal
s for
eac
h co
nditi
on o
r tim
e po
int.
Pros
pect
ive
in v
ivo
imag
ing
of d
rug
biod
istr
ibut
ion
may
pro
vide
an
alte
rnat
ive
to “c
lass
ical
” pha
rmac
oki-
netic
s and
bio
dist
ribu
tion
stud
ies i
n th
at a
con
tras
t-en
hanc
ed im
agin
g si
gnal
cou
ld se
rve
as a
surr
ogat
e fo
r the
am
ount
of d
rug
or
biol
ogic
del
iver
ed to
the
orga
n of
inte
rest
. For
the
brai
n-ta
rget
ing
appl
icat
ions
, it i
s nec
essa
ry to
dev
elop
form
ulat
ion
stra
tegi
es
that
ena
ble
a si
mul
tane
ous d
rug
and
cont
rast
age
nt d
eliv
ery
acro
ss th
e BB
B. In
this
cha
pter
, we
desc
ribe
met
hods
for e
ncap
sula
ting
drug
s int
o lip
osom
e na
noca
rrie
rs w
ith su
rfac
e di
spla
y of
bot
h th
e im
agin
g co
ntra
st a
gent
for o
ne o
r mul
tiple
imag
ing
mod
aliti
es
and
the
sing
le-d
omai
n an
tibod
y th
at u
nder
goes
rece
ptor
-med
iate
d tr
ansc
ytos
is a
cros
s the
BBB
. Con
tras
t-en
hanc
ed im
agin
g si
gnal
de
tect
ed in
the
brai
n af
ter i
ntra
veno
us in
ject
ion
of su
ch fo
rmul
atio
n(s)
is p
ropo
rtio
nal t
o th
e am
ount
of d
rug
deliv
ered
into
the
brai
n pa
renc
hym
a. Th
is m
etho
d al
low
s for
a p
rosp
ectiv
e, n
onin
vasi
ve e
stim
atio
n of
dru
g de
liver
y, ac
cum
ulat
ion,
and
elim
inat
ion
from
the
brai
n.
Iqba
l et
al. 2
011
Mim
otop
e se
lect
ion
IgN
AR
Plas
mod
ium
falc
ipar
um
Mim
otop
es m
imic
the
thre
e-di
men
sion
al to
polo
gy o
f an
antig
en e
pito
pe, a
nd a
re fr
eque
ntly
reco
gniz
ed b
y an
tibod
ies w
ith a
ffini
-tie
s com
para
ble
to th
ose
obta
ined
for t
he o
rigi
nal a
ntib
ody-
antig
en in
tera
ctio
n. P
eptid
es a
nd a
nti-
idio
typi
c an
tibod
ies a
re tw
o cl
asse
s of p
rote
in m
imot
opes
that
mim
ic th
e to
polo
gy (b
ut n
ot n
eces
sari
ly th
e se
quen
ce) o
f the
par
enta
l ant
igen
. In
this
stud
y, w
e co
mbi
ne th
ese
two
clas
ses b
y se
lect
ing
mim
otop
es b
ased
on
sing
le d
omai
n Ig
NA
R an
tibod
ies,
whi
ch d
ispl
ay e
xcep
tiona
lly lo
ng
CD
R3 lo
op re
gion
s (an
alog
ous t
o a
cons
trai
ned
pept
ide
libra
ry) p
rese
nted
in th
e co
ntex
t of a
n im
mun
oglo
bulin
fram
ewor
k w
ith
adja
cent
and
supp
ortin
g C
DR1
loop
s. B
y sc
reen
ing
an in
vitr
o ph
age-
disp
lay
libra
ry o
f IgN
AR
vari
able
dom
ains
(V(N
AR)
s) a
gain
st
the
targ
et a
ntig
en m
onoc
lona
l ant
ibod
y M
Ab5
G8,
we
obta
ined
four
pot
entia
l mim
otop
es. M
Ab5
G8
targ
ets a
line
ar tr
ipep
tide
epito
pe (A
YP) i
n th
e fle
xibl
e si
gnal
sequ
ence
of t
he P
lasm
odiu
m fa
lcip
arum
Api
cal M
embr
ane
Ant
igen
-1 (A
MA
1), a
nd th
is o
r si
mila
r mot
ifs w
ere
dete
cted
in th
e C
DR
loop
s of a
ll fo
ur V
(NA
R)s.
The
V(N
AR)
s, 1
-A-2
, -7,
-11,
and
-14,
wer
e de
mon
stra
ted
to
bind
spec
ifica
lly to
this
par
atop
e by
com
petit
ion
stud
ies w
ith a
n ar
tifici
al p
eptid
e an
d al
l sho
wed
enh
ance
d affi
nitie
s (3–
46 n
M)
com
pare
d to
the
pare
ntal
ant
igen
(175
nM
). C
ryst
allo
grap
hic
stud
ies o
f rec
ombi
nant
pro
tein
s 1-A
-7 a
nd 1
-A-1
1 sh
owed
that
the
SYP
mot
ifs o
n th
ese
V(N
AR)
s pre
sent
ed a
t the
tip
of th
e ex
pose
d C
DR3
loop
s, id
eally
pos
ition
ed w
ithin
bul
ge-li
ke st
ruct
ures
to
mak
e co
ntac
t with
the
MA
b5G
8 an
tibod
y. Th
ese
loop
s, in
par
ticul
ar in
1-A
-11,
wer
e fu
rthe
r sta
biliz
ed b
y in
ter-
and
intr
a- lo
op
disu
lphi
de b
ridg
es, h
ydro
gen
bond
s, e
lect
rost
atic
inte
ract
ions
, and
aro
mat
ic re
sidu
e pa
ckin
g. W
e ra
tiona
lize
the
high
er a
ffini
ty o
f th
e V
(NA
R)s c
ompa
red
to th
e pa
rent
al a
ntig
en b
y su
gges
ting
that
adj
acen
t CD
R1 a
nd fr
amew
ork
resi
dues
con
trib
ute
to b
indi
ng
affini
ty, t
hrou
gh in
tera
ctio
ns w
ith o
ther
CD
R re
gion
s on
the
antib
ody,
thou
gh o
f cou
rse
defin
itive
supp
ort o
f thi
s hyp
othe
sis w
ill
rely
on
co-c
ryst
allo
grap
hic
stud
ies.
Alte
rnat
ivel
y, th
e se
lect
ion
of m
imot
opes
from
a la
rge
(< 4
× 1
08 ) con
stra
ined
libr
ary
may
hav
e al
low
ed se
lect
ion
of v
aria
nts w
ith e
ven
mor
e fa
vora
ble
epita
pe to
polo
gies
than
pre
sent
in th
e or
igin
al a
ntig
enic
stru
ctur
e, il
lust
rat-
ing
the
pow
er o
f in
vivo
sele
ctio
n of
mim
otop
es fr
om p
hage
-dis
play
ed m
olec
ular
libr
arie
s.
Sim
mon
s et
al.
2008
Review Article Veterinarni Medicina, 57, 2012 (9): 439–513
504
Bac
teri
opha
ge p
2M
ajor
cap
sid
prot
ein
Rec
epto
r-bi
ndin
g
prot
eins
Neu
tral
isat
ion
assa
y
Back
grou
nd: B
acte
riop
hage
s inf
ectin
g la
ctic
aci
d ba
cter
ia (L
AB)
are
wid
ely
ackn
owle
dged
as t
he m
ain
caus
e of
milk
ferm
enta
tion
failu
res.
In th
is st
udy,
we
desc
ribe
the
surf
ace-
expr
essi
on a
s wel
l as t
he se
cret
ion
of tw
o fu
nctio
nal l
lam
a he
avy-
chai
n an
tibod
y fr
agm
ents
, one
bin
ding
to th
e m
ajor
cap
sid
prot
ein
(MC
P) a
nd th
e ot
her t
o th
e re
cept
or-b
indi
ng p
rote
ins (
RBP)
of t
he la
ctoc
occa
l ba
cter
ioph
age
p2, b
y la
ctob
acill
i in
orde
r to
neut
ralis
e la
ctoc
occa
l pha
ges.
Res
ults
: The
antib
ody
frag
men
t VH
H5
that
is d
irec
ted
agai
nst t
he R
BP, w
as fu
sed
to a
c-m
yc ta
g an
d ex
pres
sed
in a
secr
eted
form
by
a La
ctob
acill
us st
rain
. The
frag
men
t VH
H2
that
is
bin
ding
to th
e M
CP,
was
fuse
d to
an
E-ta
g an
d an
chor
ed o
n th
e su
rfac
e of
the
lact
obac
illi.
Surf
ace
expr
essi
on o
f VH
H2
was
co
nfirm
ed b
y flo
w c
ytom
etry
usi
ng a
n an
ti-E-
tag
antib
ody.
Effici
ent b
indi
ng o
f bot
h th
e V
HH
2 an
d th
e se
cret
ed V
HH
5 fr
agm
ent
to th
e ph
age
antig
ens w
as sh
own
in E
LISA
. Sca
nnin
g el
ectr
on m
icro
scop
y sh
owed
that
lact
obac
illi e
xpre
ssin
g V
HH
2 an
chor
ed a
t th
eir s
urfa
ce w
ere
able
to b
ind
lact
ococ
cal p
hage
s. A
neu
tral
isat
ion
assa
y al
so c
onfir
med
that
the
secr
eted
VH
H5
and
the
anch
ored
V
HH
2 fr
agm
ents
pre
vent
ed th
e ad
sorp
tion
of la
ctoc
occa
l pha
ges t
o th
eir h
ost c
ells
. Con
clus
ion:
Lac
toba
cilli
wer
e ab
le to
exp
ress
fu
nctio
nal V
HH
frag
men
ts in
bot
h a
secr
eted
and
a c
ell s
urfa
ce fo
rm a
nd re
duce
d ph
age
infe
ctio
n of
lact
ococ
cal c
ells
. Lac
toba
cilli
ex
pres
sing
llam
a he
avy-
chai
n an
tibod
y fr
agm
ents
repr
esen
t a n
ovel
way
to li
mit
phag
e in
fect
ion.
Hul
tber
g et
al.
2007
Prot
ein
swit
ches
Com
bina
tori
al h
isti
dine
lib
rary
pH-d
epen
dent
bin
ding
Ther
e is
gro
win
g in
tere
st in
the
deve
lopm
ent o
f pro
tein
switc
hes,
whi
ch a
re p
rote
ins w
hose
func
tion,
such
as b
indi
ng a
targ
et
mol
ecul
e, c
an b
e m
odul
ated
thro
ugh
envi
ronm
enta
l tri
gger
s. E
ffort
s to
engi
neer
hig
hly
pH se
nsiti
ve p
rote
in-p
rote
in in
tera
c-tio
ns ty
pica
lly re
ly o
n th
e ra
tiona
l int
rodu
ctio
n of
ioni
zabl
e gr
oups
in th
e pr
otei
n in
terf
ace.
Suc
h ex
peri
men
ts a
re ty
pica
lly ti
me
inte
nsiv
e an
d of
ten
sacr
ifice
the
prot
ein’
s affi
nity
at t
he p
erm
issi
ve p
H. Th
e un
derl
ying
ther
mod
ynam
ics o
f pro
ton-
linka
ge d
ic-
tate
that
the
pres
ence
of m
ultip
le io
niza
ble
grou
ps, w
hich
und
ergo
a p
K(a
) cha
nge
on p
rote
in b
indi
ng, a
re n
eces
sary
to re
sult
in
high
ly p
H-d
epen
dent
bin
ding
. To
test
this
hyp
othe
sis,
a n
ovel
com
bina
tori
al h
istid
ine
libra
ry w
as d
evel
oped
whe
re e
very
pos
sibl
e co
mbi
natio
n of
his
tidin
e an
d w
ild-t
ype
resi
due
is sa
mpl
ed th
roug
hout
the
inte
rfac
e of
a m
odel
ant
i-RN
ase
A si
ngle
dom
ain
VH
H
antib
ody.
Ant
ibod
ies w
ere
cose
lect
ed fo
r hig
h-affi
nity
bin
ding
and
pH
-sen
sitiv
ity u
sing
an
in v
itro,
dua
l-fun
ctio
n se
lect
ion
stra
tegy
. Th
e re
sulti
ng a
ntib
odie
s ret
aine
d ne
ar w
ild-t
ype
affini
ty y
et b
ecam
e hi
ghly
sens
itive
to sm
all d
ecre
ases
in p
H, d
rast
ical
ly d
ecre
as-
ing
thei
r bin
ding
affi
nity
, due
to th
e in
corp
orat
ion
of m
ultip
le h
istid
ine
grou
ps. S
ever
al tr
ends
wer
e ob
serv
ed, s
uch
as h
istid
ine
“hot
-spo
ts”,
whi
ch w
ill h
elp
enha
nce
the
deve
lopm
ent o
f pH
switc
h pr
otei
ns a
s wel
l as i
ncre
ase
our u
nder
stan
ding
of t
he ro
le o
f io
niza
ble
resi
dues
in p
rote
in in
terf
aces
. Ove
rall,
the
com
bina
tori
al a
ppro
ach
is ra
pid,
gen
eral
, and
robu
st a
nd sh
ould
be
capa
ble
of
prod
ucin
g hi
ghly
pH
-sen
sitiv
e pr
otei
n affi
nity
reag
ents
for a
num
ber o
f diff
eren
t app
licat
ions
.
Mur
-ta
ugh
et
al. 2
011
Imm
unoa
ffini
ty
chro
mat
ogra
phy
Caff
eine
Gra
ftin
g
This
wor
k de
mon
stra
tes t
he fe
asib
ility
of u
sing
a c
amel
id si
ngle
dom
ain
antib
ody
for i
mm
unoa
ffini
ty c
hrom
atog
raph
ic se
pera
tion
of sm
all m
olec
ules
. An
anti-
caffe
ine
VH
H a
ntib
ody
was
pro
duce
d by
gra
ftin
g th
e co
mpl
emen
tant
y de
term
inin
g se
quen
ces o
f a
prev
ious
ly g
ener
ated
ant
ibod
y on
to a
n an
ti-RN
ase
A a
ntib
ody
scaff
old
follo
wed
by
expr
essi
on in
E. c
oli.
Ana
lysi
s of t
he b
indi
ng
prop
ertie
s of t
he a
ntib
ody
by E
LISA
and
fluo
resc
ence
-bas
ed th
erm
al sh
ift a
ssay
s sho
wed
that
it re
cogn
izes
not
onl
y ca
ffein
e, b
ut
also
theo
phyl
line,
theo
biom
ine
and
para
xant
hine
, alb
eit w
ith lo
wer
affi
nity
. Fur
ther
inve
stig
atio
n of
the
effec
t of e
nvir
onm
enta
l co
nditi
ons,
i.e.
, tem
pera
ture
, pH
, and
ioni
c st
reng
th, o
n th
e an
tibod
y us
ing
thes
e m
etho
ds p
rovi
ded
usef
ul in
form
atio
n ab
out
pote
ntia
l ela
tion
cond
ition
s to
be u
sed
in c
hrom
atog
raph
ic a
pplic
atio
ns. I
mm
obili
zatio
n of
the
VH
H o
nto
a hi
gh fl
ow-t
hrou
gh
synt
hetic
supp
ort m
ater
ial r
esul
ted
In a
stat
iona
ry p
hase
cap
able
of s
epar
atin
g ca
ffein
e an
d its
met
abol
ites.
Fran
co e
t al
. 201
0
Veterinarni Medicina, 57, 2012 (9): 439–513 Review Article
505
Prot
ein
conf
orm
atio
nSp
ectr
al p
rope
rtie
sG
reen
fluo
resc
ent
prot
ein
Prot
ein
tran
sloc
atio
n
Prot
ein
conf
orm
atio
n is
cri
tical
ly li
nked
to fu
nctio
n an
d of
ten
cont
rolle
d by
inte
ract
ions
with
regu
lato
ry fa
ctor
s. H
ere
we
repo
rt
the
sele
ctio
n of
cam
elid
-der
ived
sing
le-d
omai
n an
tibod
ies (
nano
bodi
es) t
hat m
odul
ate
the
conf
orm
atio
n an
d sp
ectr
al p
rope
rtie
s of
the
gree
n flu
ores
cent
pro
tein
(GFP
). O
ne n
anob
ody
coul
d re
vers
ibly
redu
ce G
FP fl
uore
scen
ce b
y a
fact
or o
f 5, w
here
as it
s dis
plac
e-m
ent b
y a
seco
nd n
anob
ody
caus
ed a
n in
crea
se b
y a
fact
or o
f 10.
Str
uctu
ral a
naly
sis o
f GFP
-nan
obod
y co
mpl
exes
reve
aled
that
the
two
nano
bodi
es in
duce
subt
le o
ppos
ing
chan
ges i
n th
e ch
rom
opho
re e
nvir
onm
ent,
lead
ing
to a
ltere
d ab
sorp
tion
prop
ertie
s. U
nlik
e co
nven
tiona
l ant
ibod
ies,
the
smal
l, st
able
nan
obod
ies a
re fu
nctio
nal i
n liv
ing
cells
. Nan
obod
y-in
duce
d ch
ange
s wer
e de
tect
ed b
y ra
tio im
agin
g an
d us
ed to
mon
itor e
vent
s suc
h as
the
tam
oxife
n-in
duce
d nu
clea
r loc
aliz
atio
n of
est
roge
n re
cept
or. Th
is w
ork
dem
-on
stra
tes t
hat p
rote
in c
onfo
rmat
ions
can
be
man
ipul
ated
and
stud
ied
with
nan
obod
ies i
n liv
ing
cells
.
Kir
ch-
hofe
r et
al. 2
010
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Received: 2012–08–30Accepted after corrections: 2012–09–25
Corresponding Author:
Mgr. Ludek Eyer, Ph.D., Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech RepublicTel. +420 533 331 911, E-mail: eyer@vri.cz
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