Characterization of Physical Chemical Properties of
Nanobody Against Helicobacter Pylori Urease
L. Safaee Ardekani1, SL. Mousavi Gargari1, M.Rajabi Bazl2, W.Ebrahimizadeh1
1Department
2Department
of biology, faculty of basic science, shahed university, Tehran, Iran
of biochemistry,faculty of medical science, beheshti university, Tehran, Iran
Key words:Nanobody, H.pylori, physical Chemical Test
leilasafaee@yahoo.com
Introduction
Antibodies or antibody fragments are almost exclusively applied in human therapy or diagnosis . Antibodies high
specificity makes them suitable in many applications and processes (1). Recently, as a new effective therapeutic strategy,
treatment of H. pylori infection has been studied by antibody administration against urease enzyme (2). Despite
widespread acceptance and promises, classic antibodies still have functional limitations including large size, interaction
with patient immune system, inadequate pharmacokinetics and tissue accessibility (3). Heavy chain antibodies (HCAbs)
which are a type of antibodies that lack the light chain and CH1 domain were discovered in serum of camelids in early
1990s. These antibodies have a single variable domain that referred to as VHH, sdAb or nanobody. Nanobodies, with
small size (2.5nm in diameter and nearly 4 nm high) (4), have better tissue penetration and effective pharmacodynamics
while they have less interference with the host immune system . Moreover, other Significant advantages such as, high
stability against extreme temperature and pH , physical stability, capability of refolding and recognition of unique epitopes
that are inaccessible to conventional antibodies (5,6) give them more therapeutic value compared to conventional
antibodies and antibody fragments. The present study was aimed at production of nanobody against UreC recombinant
protein and evaluation of physical - chemical properties.
1. Material and methods
1. 1. In vitro proteolytic stability of VHHs
Proteolytic stability of VHHs was analyzed using porcine pepsin and bovine trypsin. Five and ten μg/ml of purified
nanobody were incubated with different concentration of pepsin in10 mM HCl (pH 2) and trypsin in 1 mM Tris–Cl and 20
mM CaCl2 (pH 8. 0) 1h at 37°C prior to the test. For control test, PBS was used instead of Proteolytic enzymes. The
affinity of nanobody was determined by antigen-specific ELISA. Polystyrene 96-well plates were coated with 5 and 10
μg/ml UreC in 0.05 M carbonate/bicarbonate buffer, pH 9. 6. Residual sites were blocked for 1h with 0.5% skimmed milk
in PBS. After washing with PBS-T, incubated nanobodies in proteolytic enzymes were added to the wells and kept for 2h
at 37o C. Following wash with PBS-T, HRP-conjugated anti-His antibody was added at the final concentration of 1.10000
to the wells and the plate was incubated 1 h at 37o C. Immunoreactivity was developed with TMB substrate. The reaction
was stopped with H2SO4 (3N) and the OD was measured at 450 nm.
1. 2. Temperature treatment
5 and 10 μg/ml of Nanobody in PBS was incubated for 2 h at various temperatures (4³C, 25³C, 60³C, 80³C, 90³C),
followed by 30 min incubation at room temperature (RT) and then stored at 4³C. ELISA was developed as described
under “In vitro proteolytic stability of VHHs”. (7)
1. 3. Binding in the presence of urea
Different concentrations of nanobody (5 and 10 μg/ml) were incubated overnight at room temperature in 0-8 M of Urea in
PBS. ELISA was carried out as mentioned before using Urea treated nanobodies.(7)
2. Results:
2. 1. Proteolytic stability
Proteolytic stability of nanobody was studied after incubation with different concentrations of purified pepsin and trypsin.
As illustrated in Figure 1 the nanobodies retained their full activity even when incubated with high concentration of
proteolytic enzymes.
2. 2. Temperature stability
In order to investigate the temperature stability, nanobodies were incubated at a range of temperatures.
As shown in Figure 2a, nanobodies preserved functional even after incubation at 90³C. Figure 2a demonstrates that
antigen binding in nanobodies is unaffected by the temperature.
2. 3. Binding in the presence of Urea
Nanobody has preserved binding when tested in an ELISA experiment in the presence of increasing amounts of Urea as
shown in Figure 2b. This result suggests that the urea treated VHHs retained function and antigen binding ability even in
denaturing conditions.
3. Discussion
Nanobody is a potential interest for usage in applications other than diagnostics and human therapy. Since little is known
in respect to affinity, specificity and stability of such fragments we aimed to investigate these aspects. Iin this study,
specific nanobody to UreC antigen was investigated under various conditions. Urea solution was used to investigate
whether that nanobody could preserve antigen binding in a denaturating condition. Denaturing condition provided method
for characterizing the stability of nanobody and function in unfolded state.
Our results show that our nanobody preserve antigen binding in the presence of Urea. Complete recovery of the
biological activity after renaturation demonstrates that chemical-induced unfolding is fully reversible in these antibody
fragments. The effectiveness of oral immunotherapy is often complicated by partial proteolytic degradation of antibodies
within the gastro-intestinal (GI) tract. Proteolysis of ingested proteins is initiated by the action of pepsin in the stomach
and continued in the duodenum by trypsin. Use of engineered recombinant antibody fragments which are resistance to
proteolysis opens new approach for oral immunotherapy. Our results showed UreC specific nanobody remained intact at
high concentrations of pepsin and trypsin, therefore this nanobody is a promising candidate for oral immunotherapy
against H. pylori infection. In another experiment nanobody was tested for functional binding at high temperatures.
Therefore we propose that heat stability may also be explained from successful refolding of nanobody after heat
denaturation . Our results indicated that nanobody specifically binds to UreC in temperatures as high as 90 C.Such high
thermodynamic stability has never been reported for any functional conventional antibody fragment, even when
engineered antigen binders are considered. Hence, the reduced size, improved solubility, and higher stability of
nanobodies are of special interest for biotechnological and medical applications.
This particular property extends the use of nanobody dramatically. For example, nanobody can be used in processes in
which a high temperature step (pasteurization) is involved, without losing the ability of antigen binding. These properties
give nanobody in (heat) denaturing conditions an advantage over more complex antibodies such as mouse mAbs and
scFvs.
Fig1: a: Proteolytic stability of nanobody after incubation with trypsin proteases. b: Proteolytic stability of nanobody after
incubation with pepsin proteases.
Fig. 2. a: Antigen binding after temperature treatment of nanobody. Nanobody is incubated for 2 h at different
temperatures, cooled down to room temperature and subsequently ELISA was performed. b: Antigen binding of VHH after
overnight incubation with Urea. Consistence of antigen binding in different concentration of Urea is observed suggesting
that VHH function was not affected by Urea.
References :
1. Hamers-Casterman, C., T. Atarhouch, et al. "Naturally occurring antibodies devoid of light chains." Nature
,1993;363(6428): 446-448
2. WU, Y., Q. ZOU, et al. "Preparation of anti-Helicobacter pylori UreB monoclonal antibody and the detection of the
ability of inhibition the urease activity [J]." Immunological Journal , 2005
3. Reiche, N., A. Jung, et al. "Generation and characterization of human monoclonal scFv antibodies against
Helicobacter pylori antigens." Infection and immunity, 2002; 70(8): 4158.
4. Chames, P., M. Van Regenmortel, et al. "Therapeutic antibodies: successes, limitations and hopes for the future."
British Journal of Pharmacology,2009; 157(2): 220-233.
5. Spinelli, S., L. G. J. Frenken, et al. "Camelid heavy-chain variable domains provide efficient combining sites to
haptens." Biochemistry , 2000; 39(6): 1217-1222.
6. Alvarez-Rueda, N., M. Z. Ladjemi, et al. "A llama single domain anti-idiotypic antibody mimicking HER2 as a
vaccine: Immunogenicity and efficacy." Vaccine, 2009; 27(35): 4826-4833
7. Van der Linden, R., L. Frenken, et al. "Comparison of physical chemical properties of llama VHH antibody
fragments and mouse monoclonal antibodies." Biochimica et Biophysica Acta (BBA)-Protein Structure and
Molecular Enzymology , 1999; 1431(1): 37-46