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Iranian Journal of Basic Medical Sciences
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Preparation and characterization of a novel nanobody against
T-cell immunoglobulin and mucin-3 (TIM-3)
Vida Homayouni 1, Mazdak Ganjalikhani-hakemi 1*, Abbas Rezaei 1, Hossein Khanahmad 2,
Mahdi Behdani 3, Fatemeh Kazemi Lomedasht 3
1 Immunology Department, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
2 Genetic Department, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
3 Biotechnology Research Center, Biotechnology Department, Venom & Bio-therapeutics Molecules Lab, Pasteur Institute of Iran, Tehran, Iran
A R T I C L E I N F O
A B S T R A C T
Article type:
Original article
Objective(s): As T-cell immunoglobulin and mucin domain 3 (TIM-3) is an immune regulatory
molecule; its blocking or stimulating could alter the pattern of immune response towards a
desired condition. Based on the unique features of nanobodies, we aimed to construct an anti-TIM-
3 nanobody as an appropriate tool for manipulating immune responses for future therapeutic
purposes.
Materials and Methods: We immunized a camel with TIM-3 antigen and then, synthesized a VHH
phagemid library from its B cell’s transcriptome using nested PCR. Library selection against TIM-
3antigen was performed in three rounds of panning. Using phage-ELISA, the most reactive
colonies were selected for sub-cloning in soluble protein expression vectors. The Nanobody was
purified and confirmed with a nickel-nitrilotriacetic acid (Ni-NTA) column, SDS-PAGE and Western
blotting. A flowcytometric analysis was performed to analyze the binding and biologic activities of
theTIM-3 specific nanobody with TIM-3 expressing HL-60 and HEK cell lines.
Results: Specific 15kD band representing for nanobody was observed on the gel and confirmed
with Western blotting. The nanobody showed significant specific immune-reactivity against TIM-3
with a relatively high binding affinity. The nanobody significantly suppressed the proliferation of
TIM-3 expressing HL-60 cell line.
Conclusion: Finally, we successfully prepared a functional anti-humanTIM-3 specific nanobody
with a high affinity and an anti-proliferative activity on an AML cell line in vitro.
Article history:
Received: May 15, 2016
Accepted: Jun 30, 2016
Keywords:
Antibody
Heavy chain antibody
Nanobody
Phage display
T-cell immunoglobulin and -
mucin domain 3
►
Please cite this article as:
Homayouni V, Ganjalikhani-hakemi M, Rezaei A, Khanahmad H, Behdani M, Kazemi Lomedasht F. Preparation and characterization of a
novel nanobody against T-cell immunoglobulin and Mucin-3 (TIM-3).
Iran J Basic Med Sci 2016; 19:1201-1208.
Introduction
The human TIM family members (TIM-1, TIM-3,
TIM-4) are commonly expressed as surface
glycoproteins containing an IgV domain, a mucin stalk,
a transmembrane domain, and a cytoplasmic tail (1, 2).
The gene encoding TIM-3 is known as HAVCR2, which
denotes hepatitis A virus (HAV) cellular receptor 2 (3).
TIM-3 has important and complex roles in the
regulation of immune responses. TIM-3 was originally
identified as surface molecule on CD4 effector Th1 cells
(4, 5). Now, it is known that TIM-3 is also expressed on
dendritic cells, microglia, macrophages, mast cells; NK
cells, activated and exhausted CD8 T cells (4, 6, 7).
The exhausted CD8T cells are generated as a
consequence of prolonged immune responses to
chronic infections and tumors (8). These cells are
characterized by a failure to proliferate and to exert
their effector functions such as cytotoxicity and
cytokine secretion in response to antigen stimulation
(9). Stimulation of TIM-3 by its ligand, galectin-9,
results in Th1 cell death, implicating a role for TIM-3 in
negatively regulating Th1 responses. Blocking of TIM-3
has been shown to increase IFN-γ secreting T cells,
mediating the pathophysiology of Th1-driven
autoimmune diseases. TIM-3 expressed on
macrophages and monocytes is implicated in
phagocytosis of apoptotic cells and cross-presentation
(10). In tumors, TIM-3 expression is induced on innate
immune cells, leading to a suppression of innate
responses to nucleic acids released from apoptotic
tumor cells by interacting with HMGB1. As discussed,
TIM-3 interacts with galectin-9 (Gal 9) and causes
exhaustion and apoptosis of antigen-specific Th1 cells
and CTLs, which correlates with impaired antitumor
immune response (11). Blocking of TIM-3 interaction
with its ligand would restore effector functions of
exhausted CD8 T cells and its anti-tumor activity (12-
14). Therefore, TIM-3 blockade and Treg depletion
could have a synergistic effect on tumor growth
inhibition (15).
*Corresponding author: Mazdak Ganjalikhani-hakemi. Immunology Department, Faculty of Medicine, Isfahan University of Medical Sciences. Isfahan, Iran.
Tel: +98-31-37929082; Fax: +98-31-3792903 1; email: mghakemi@med.mui.ac.ir
Homayouni et al Novel Nanobody prepared against TIM-3
Iran J Basic Med Sci, Vol. 19, No.11, Nov 2016
1202
TIM
-3 is also expressed on cancer stem cells in patients
with acute myeloid leukemia (AML) and has a role in
promoting myeloid-derived suppressor cells (MDSC).
Furthermore, in AML cell lines (HL-60), TIM-3 moderately
activates the mammalian target of rapamycin (mTOR, a
master regulator of myeloid cell translational pathways).
This results in activation of the hypoxia-inducible factor 1
(HIF-1) transcription complex
, which upregulates
glycolysis and expression/secretion of the pro-
angiogenic vascular endothelial growth factor (VEGF)
(16). In a study, anti-human TIM-3 antibody blocked
AML engraftment in a xenotransplant model (17).
The role of TIM-3 in modulation of autoimmune
diseases has been studied in experimental autoimmune
encephalomyelitis (EAE), as a mouse model of MS. Low-
level expression of TIM-3 on EAE Th1 cells may explain
their Th1 resistance to inhibition induced by TIM-3/
galectin-9 pathway (18).
Now a day, antibodies are widely used as diagnostic
and therapeutic tools in clinic. The large size of
monoclonal antibodies (mAb) in some clinical
situations is a drawback, which limits their penetration
and distribution deep into tissues (19). In addition, the
Fc portion of mAbs is more likely to activate
complement system or phagocytosis process in vivo
rather than having solely a blocking or stimulating
activity. To overcome these limitations, fragments of
antibodies have been generated. Among antibody
fragments, nanobodies (Nbs) have unique features,
which make them appropriate tools for manipulating
immune responses for therapeutic purposes (20, 21).
This unique type of antibody is found in sera of camels
(dromedaries) and llamas. These camelid antibodies
lack light chains and are called heavy-chain antibodies
(HcAbs). The variable domain of HcAbs is named VHH,
Nanobody or single domain antibody, which is
responsible only for antigen binding. Nbs are the
smallest fully functional antigen-binding fragment of
these antibodies. It appears that Nbs have very low
immunogenicity for human because of a high degree of
sequence similarity between VHH and human VH
sequences (22, 23). Nbs are more efficient in
immunotherapy, because they have small size, which
enable them to access inaccessible positions in the
body. They also have high affinity and specificity for
their cognate antigen. Based on the importance of
blocking and/or stimulating of TIM-3 signaling in
different pathologic conditions, this study was aimed to
design and produce a monoclonal Nb with a high
affinity against the TIM-3 protein for further in vitro
and in vivo studies.
Materials and Methods
Preparation of antigen for immunization
The HEK 293 cell line that expressed recombinant
human TIM-3 was prepared for immunization (in
press). A 6 months Camelus dromedaries was
intramuscularly immunized with 50 μg purified Human
TIM-3 Protein (Sinobiological, inc) plus complete
Freund’s adjuvant (CFA, CMG company) followed by the
adjuvant free lysates prepared from 5×107 human TIM-
3 expressing cells for 4 times every 2 weeks. Whole
blood was collected before the first injection and 7 days
after each injection, and the sera were isolated for
measurement of the antibody titer.
Library construction
Ten days after the last immunization, 400 ml blood
was collected and peripheral blood mononuclear cells
(PBMC) were separated using Lymphoprep (Greiner
Bio-one). Total RNA was extracted from isolated PBMCs
with the RNX Plus reagent (Cinnagen, Iran) and cDNA
was synthetized with a RevertAid First Strand cDNA
Synthesis Kit (Fermentas, Germany) using OligodT
primer. Nested PCR was done for VHH amplification
using specific primers. The leader-specific primer
CALL001 (5′-GTC CTG GCT GCT CTT CTA CAA GG-3′)
and CH2-specific primer CALL002 (5′ -GGT ACG TGC
TGT TGA ACT GTT CC-3′) were used for VH and VHH
amplification.
PCR products were electrophoresed on a 1%
agarose gel and the 600-700 bp fragments (VHH-CH2
without CH1 exon) were purified from the gel with
AccuPrep Gel Purification Kit (Bioneer, Korea). Purified
bands were re-amplified using nested primers A6E
(5′-GAT GTG CAG CTG CAG GAG TCT GGR GGA GG-3′)
and primer 38 (5′-GGA CTA GTG CGG CCG CTG GAG
ACG GTG ACC TGG GT-3′) for framework 1 and
framework 4 regions. The pHEN4 phagemid vector and
the amplified PCR product were digested with PstI and
NotI restriction enzymes and then, ligated with T4 DNA
ligase enzyme. Recombinant vector was transformed
into electro-competent Escherichia coli TG1 cells.
Colony PCR was done to confirm the successful cloning.
Enrichment of the VHH library
The VHH libraries were displayed on phages after
their infection with VCSM13 helper phages. This library
was grown in 330 ml 2xTY media containing 100 μg/ml
ampicillin and 4% of glucose. Bacteria at mid-log phase
(OD=0.5 at 600 nm) were infected with 2×1012 CFU
of VCSM13 helper phage. Infected bacteria were
incubated for 30 min at 37 °C. After a centrifugation, the
resulted pellet was cultured in 2×TY supplied with
50 mg/ml Kanamycin and incubated at 37 °C for 16 hr
while shaking at 250 rpm. The culture medium was
centrifuged for 20 min at 9000 rpm at 4 °C and the
supernatant was mixed with polyethylene glycol (PEG)-
NaCl. This was incubated for 60 min on ice. After
centrifugation for 15 min at 4000 rpm and 4 °C, the
pellet was isolated and re-suspended in 1 ml of PBS.
Enrichment of the specific phage was done with three
rounds of in vitro selection on microtiter plates coated
with 10 μg recombinant TIM-3 protein. After each
selection round, binders were eluted with 100 mM
triethylamine (pH 10) and immediately neutralized
Novel nanobody prepared against TIM-3 Homayouni et al
Iran J Basic Med Sci, Vol. 19, No. 11, Nov 2016
1203
with 1M Tris–HCl, pH 8. Phage particles were finally
used to infect exponentially growing E. coli TG1
bacteria.
Polyclonal phage ELISA, applying the extracted
phages, was used for evaluation of the panning process
after each round. After three rounds of panning,
individual colonies of third round were randomly
picked for periplasmic extract ELISA (PE-ELISA) in
order to detect positive clones. In PE-ELISA expression
of soluble periplasmic VHHs was induced with 1 mM
isopropyl-d-1-thiogalactopyranoside (IPTG). The
periplasmic proteins were extracted using osmotic
shock with TES buffer and extracted material
containing recombinant VHH was tested for antigen
recognition.
Expression and characterization of VHH Nanobody
After PE-ELISA, samples with the highest optical
density (OD) were selected for further experiments.
The VHH genes of the selected colonies were sub-
cloned into the pHEN6C expression vector with BstEII
and PstI restriction sites. This vector includes a C-
terminal His-tag. The recombinant pHEN6C was
transformed into E. coli WK6 (Pasteur Institute, Iran)
and expressed Nb was obtained. Briefly, the
periplasmic crude extract proteins were prepared using
osmotic shock and loaded on a His-Select column
(Sigma-Aldrich, Germany). After washing with PBS, the
bound proteins were eluted with 500 mM imidazole.
SDS-PAGE and Western blotting
The purity and identity of the protein was evaluated
on a15% gel with SDS–PAGE method. The gel was
stained with Coomassie brilliant blue. Western blotting
was done with anti-His tag antibody (Abcam, UK). For
Western blotting, the gel was run at a constant voltage
of 100 V for 45 min using a Mini Protein Tetra System
(Bio-Rad, USA), in order to transfer the protein bands to
the nitrocellulose membrane (CMG Company). The
nitrocellulose membrane was then blocked with 5%
skimmed milk in PBS-T for 16 hr at 4°C. The membrane
was washed and detection was done with 1/5000
dilution of HRP conjugated anti-His Tag antibody
(Abcam, UK) and diaminobenzidine (DAB) (CMG
Company) as substrate.
Measurement of TIM-3 Nanobody affinity
Affinity of the purified Nanobodies (Nb94 and
Nb60) was determined using ELISA method. TIM-3
antigen (HAVCR2 Protein /Sino Biological Inc.) was
coated in a 96 well microplate wells (Nunc, Denmark)
in different concentrations (5 and 10 μg/ml). After
washing and blocking, the Nb was added at 0.001, 0.01,
0.1, 1, 10 nM concentrations. HRP-conjugated anti-His
antibody (Abcam, UK) was added and the immune
reactivity was assessed with TMB substrate. The
reaction was stopped with a stop solution and OD was
measured at 450 nm using a microplate reader
(Hyperion).
Flowcytometric analysis
The TIM-3 expressing HL-60 cells (an AML cell line),
induced with PMA, and TIM-3 expressing HEK 293
cells, stably transfected, as well as TIM-3 negative
HEK293 cells (as negative control) were used
for flowcytometric analysis of anti-TIM-3 Nb
immunoreactivity. About 5×105 cells were washed
three times and re-suspended in a total volume of 100
μl PBS. One microgram of Nb was added, and cells were
incubated for 45 min on ice. After washing with PBS,
cells were incubated with 1 μg mouse anti-His-tag
antibody-PE (Biolegend, UK) for 1 hr on ice. Anti-TIM-
3-PE monoclonal antibody (mAb) (Biolegend, UK) was
applied as positive control for another group of cells.
Cells were washed with PBS and analyzed with Cell
Quest Pro software in a FACS Callibour (BD Biosciences,
USA) instrument.
CFSE Proliferation assay
In the assay, HL-60 cells were grown in RPMI
medium, induced with PMA and labeled with
10 mM carboxyfluorescein-succinimidyl ester (CFSE)
(Biolegend, UK) at 37 °C for 20 min in the dark and
washed twice with PBS containing 10% FBS to remove
excessive CFSE. Cells were seeded at 2.5×105 cells/well
in 24-well plates and incubated at 37 °C with 5% CO2. At
least 24 hr before analysis, 2 μl (1 μM) galectin-9
(Biolegend, UK) was added into each well. One group
was treated with our anti-TIM-3 Nanobody (10 µl) as
the test group and a standard anti-TIM-3 mAb (10 µl)
was added to another group as the positive control. One
group of the seeded cells was not treated with galectin-
9 as the negative control. At the appropriate point in
time, cells were washed twice, re-suspended in PBS
buffer, and analyzed immediately using a FACS Calibur
flowcytometer (Becton Dickinson, USA).
Statistical analysis
Paired-T-Test and one way ANOVA were applied to
compare the results before and after interventions
between two and more than two groups, respectively.
P<0.05 was considered as the statistical significance. All
the analyses were done using SPSS 20 (SPSS Inc.,
Chicago, IL, USA).
Table 1. Camel anti-TIM-3 antibody level after each round of
immunization
Test
Control
(Non specific binding)
Pre-immunization
0.149±0.01
0.19±0.05
Final immunization
1.7±0.2
0.141±0.04
Homayouni et al Novel Nanobody prepared against TIM-3
Iran J Basic Med Sci, Vol. 19, No.11, Nov 2016
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Figure 1. A. Library construction. Analysis of the first PCR product
with agarose gel electrophoresis (1%); the PCR product with different
sizes 900, 700, and 600 bp, M shows: molecular weight marker (1 kb).
B. Analysis of the second PCR product by gel electrophoresis (1%); the
VHH fragment with 400 bp size is showed. M, molecular weight
marker (1 kb). The a band shows 1000 bp.The b band shows 700 bp.
The c band shows 400 bp
Figure 2. Colony PCR analysis of colonies from the library. The most of
the colonies had an inserted fragment of VHH gene (600 bp). M,
molecular weight marker (100 bp)
Results
Immunization and construction of Nanobody library
Anti-human TIM-3 antibody level was increased
significantly in the serum of immunized camel
(P=0.0001) 10 days after the last injection (Table 1).
Very low optical density in controls in compare with
tests (P=0.0001) confirmed no non-specific binding
(NSB) in the experiment. Then, lymphocytes were
isolated from anti-coagulated blood of the immunized
camel. Total RNAs were purified and cDNA was
synthesized. The variable domains of the heavy-chain
antibodies were amplified with PCR and bands related
to both classical antibody (900 bp) and heavy chain
antibody (600-700 bp) were obtained (Figure 1A).
In nested PCR, a 400 bp region between the
framework 1 and framework 4 (VHH) was amplified
(Figure 1B). The digested PCR fragments were ligated
into pHEN4 phagemid vector and transformed into E.
coli TG1. The obtained library of transformant has
2.1×107 members. Colony PCR analysis of 25 randomly
picked colonies from the library showed that the most
of the colonies had a phagemid containing an inserted
fragment of VHH gene (Figure 2).
We identified TIM-3 Nbs by bio-panning in three
rounds to validate the quality of the library using phage
display technology. In ELISA enrichment, we obtained a
relatively enriched phage eluted from wells coated with
TIM-3 protein. The third round of panning showed the
highest immuno-reactivity in the polyclonal phage
ELISA (Figure 3). 48 clones were screened from the
third round of panning that bound to TIM-3
recombinant protein using PE-ELISA. The Nb49and
Nb60 clones showed the highest color intensity at 450
nm by ELISA reader and selected for expression (Figure
4).
Figure 3. The panning process with phage ELISA. The absorbance and
enrichment against TIM-3 was increased and the highest enrichment
was obtained in the third panning round. The absorbance against skim
milk (negative control) remained constant
Figure 4. Periplasmic extract (PE)-ELISA for selecting the most
secretory anti-TIM-3 VHH expressing colonies. A selected nanobody
clone showing the highest optical density at 450 nm in compare with
negative control. Presented data are mean±SD of three identical
repeats of the same experiment. Asterisks (*) shows statistical
significance compared with control
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Iran J Basic Med Sci, Vol. 19, No. 11, Nov 2016
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Expression of soluble VHH Nanobody
For expression of the binder Nbs, Nb49 and Nb60
were sub-cloned into a pHEN6C expression vector. Nb
was expressed in fusion with N-terminal His-tag and
purified by NTA-affinity chromatography, with a 0.005
μg concentration.
Nanobody Characterization
Using SDS-PAGE analysis, a single 15 kDa band
representing for the presence of the purified Nb (VHH)
was observed on polyacrylamide gel. No contamination
or degraded product was detected. Western blotting
confirmed the identity of the 15 kDa band (Figure 5).
Then, using ELISA method and Beaty equation, the
affinity of the Nb was calculated as 6×10-8 M.
A flowcytometric analysis was performed to access
the specific binding activity of the Nanobody to human
TIM-3 expressed on cell surface, compared with a
standard anti-TIM-3 antibody. Based on the obtained
results, the frequency of TIM-3 positive HEK293 cells
detected with our Nb was almost similar with the
standard anti-TIM-3 antibody (80% and 85%,
respectively). The results for HL-60 cells were also
similar (53% detected with the Nb and 65%with the
standard antibody) (Figure 6).
CFSE proliferation Assay
Engagement of TIM-3 on HL-60 cells with galectin-9
leads to the proliferation of the HL-60 cells. Here, using
PMA, HL-60 cells were induced to express TIM-3 and
then were treated with galectin-9 as a specific TIM-3
ligand. The cells proliferation was significantly
increased 24 hr after treatment with galectin-9
compared with un-treated (negative) control
(P=0.0001). Meanwhile, the proliferation of TIM-3
expressing HL-60 cells was significantly inhibited
(P=0.0001) using both the Nb and the commercial
antibody versus control (17.2% and16.3% versus
100%, respectively) (Figure7).
Figure 5. SDS-PAGE and Western blotting. A) SDS-PAGE analysis
of Nanobody purified by immobilized metal affinity
chromatography (IMAC); lane M: molecular weight marker; lane 1:
Nb49 and 2: Nb60 (TIM-3- Nanobody) eluted by 500 mM
imidazole buffer. B) Lanes 1 and 2: the specific reaction of the
HRP-conjugated anti-His Tag antibody with Nb49, Nb60,
respectively. Lane M: molecular weight marker
Figure 6. Flowcytometry analysis. A) HEK293 stably transfected
cell line, express in 85% TIM-3 stained with anti-TIM-3PE .B)
HEK293 stably transfected cell line, express in 80% TIM-3 stained
with anti-TIM-3 nanobody, mouse anti-His PE-conjugated
antibody. C) HL-60 cell line that induce for TIM-3 expression and
stained with anti-TIM-3 PE. About 65% of cells showed expressed
TIM-3.D) HL-60 cell line that induced for TIM-3 expression and
stained with anti-TIM-3 Nanobody and anti-His PE conjugated
antibody. About 53% of cells showed expressed TIM-3
Figure 7. Nanobody TIM-3 induced inhibition of HL-60 cell line
proliferation. TIM-3 expressing HL60 cells were treated with
Galectin-9 to be induced for proliferation. A) Proliferation
inhibition of HL-60 cells using anti-TIM-3 antibody. B)
Proliferation inhibition of HL-60 cells using anti-TIM-3 nanobody.
In the histograms, (1) represents for HL-60 proliferation; (2)
represents for HL60 cells were treated with Galectin-9; and (3)
represents for Proliferation inhibition. C) More than 80%
inhibition of proliferation was observed using both the antibody
and the Nanobody. No meaningful difference was observed
between the antibody and the Nanobody inhibitory effect
(P˃0.05). Asterisks (*) shows statistical significance compared
with control
Homayouni et al Novel Nanobody prepared against TIM-3
Iran J Basic Med Sci, Vol. 19, No.11, Nov 2016
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Discussion
TIM-3 exerts various important roles in the
regulation of T cell responses and innate immune
system. It has three known ligands including Galectin-9,
Phosphatidyl Serin (PS), and high-mobility group
protein 1 (HMGB-1). The interaction of Galectin-9-TIM-
3 in TIM-3-expressing Th1 cells leads to cell death. PS
binding by TIM-3 can mediate uptake of apoptotic cells
by phagocytes. Engagement of TIM-3 by HMGB-1 has
been shown to inhibit activation of resident DCs in the
tumor micro-environment (24, 25). More recently, it
has been shown that TIM-3 is present at the immune
synapse and can be colocalized with phosphatases that
suppress TCR signaling. TIM-3 has also been shown to
inhibit innate immune cells functions like IL-12
secretion and TLR-mediated activation (26, 27). Based
on these important functions in immune responses,
TIM-3 is now considered as a promising therapeutic
target for different pathologic conditions.
In the context of infectious diseases, one study has
shown that the blockade of TIM-3 engagement with its
ligands in mouse models of chronic infections can
augment T cell responses, which helps to eradicate the
infection (28). Independent reports have shown that
specific blocking of TIM-3 and PD-1 signaling pathways
improves T cell responses leading to control of viral
infection in chronically infected patients (14, 24). In a
recent study, it has been reported that, in the presence
of TIM-3 blocking antibodies, the production of IL-6,
IFN-γ and TNF-α is increased in Mycobacterium
tuberculosis and in viral infections, which was very
useful in inducing macrophage activation and
restricting microbial growth (28).
Several blocking mAbs have been generated to
target mouse and human TIM-3, so far. These include
RMT3-23, 8B.2C12, 2E2, AF2365, ATIK2a, 344823 and
344801 which differentially affect immune functions
(14, 19, 29, 30). RMT3–23 is an anti-mouse TIM-3
blocking mAb and has been widely used and tested in
various murine tumor models including melanoma,
colon adenocarcinoma, sarcoma, prostate carcinoma,
and ovarian cancer. Immunotherapy with RMT3–23
mAb alone was highly effective to increase tumor-
infiltrating IFN-γ-producing CD4 and CD8 T cells and to
suppress T regulatory (Treg) functions in sarcoma and
colon adenocarcinoma models (30-32).
TIM-3 also is being considered as a target antigen
for anti-leukemia therapy including T-cell leukemia and
AML as well as for reversing T cell exhaustion and
restoring anti-tumor immunity. Therefore, the success
in TIM-3 blockade could be a major step forward to the
development of immunotherapy for the treatment of
different diseases such as cancers (33).
Inaccessible anatomical points are a bottleneck for
obtaining acceptable clinical outcome after intervention
with mAbs, as some therapeutic monoclonal antibodies
need to penetrate deep into such tissues to reach their
targets. In order to facilitate Ab accession to
inaccessible points, Ab fragments such as Scfv, Fv, Fab
and HcAbs have been introduced, so far.
HcAbs can overcome this bottleneck as they include
only a single variable domain (called VHH from camel
Ab), which generates high affinities towards a large
spectrum of antigens. These small domains (15 kDa)
can be easily produced in bacteria or yeasts and are
then called domain antibodies (dAbs), or Nbs (34). Nbs
are clearly privileged compared with conventional
antibody fragments. Some reports of the use of Nbs
against tumor associated cell-surface markers have
shown that Nbs can act like normal antibodies (35-37).
Heavy chain antibodies (VHH) have provided new
opportunities in clinical applications of mAbs. These
unique antibodies interact with the antigen by virtue of
only one single variable domain. The recombinant Nbs
selected from phage display libraries are well
expressed, highly soluble in aqueous environments,
very robust and have high sequence homology with the
human variable region gene family (23).
The affinity of different antibodies has been
previously reported in the range of 10-7 to 10-10M (38).
In the present work, the equilibrium dissociation
constant (Kd) of the anti-TIM-3 Nb was determined as
6×10 -8 M which is a suitable affinity. Several studies
shown that very high affinities can be suboptimal for
therapeutic antibodies that target solid tumors. As high
affinity antibodies tightly bind their specific antigen
upon the first encounter, at the periphery of the tumor,
they do not penetrate deeper inside the tumor until all
antigen molecules are saturated at the periphery. By
contrast, moderate binders are released from these first
encountered antigens and penetrate deeper into the
tumor, ultimately leading to uniform intratumoral
distribution and higher tumor uptake (34). VHHs,
unlike conventional antibodies, can recognize epitopes
in the inaccessible sites such as caves and clefts; hence,
penetrate more deeply in different sites (34).
Roovers et al have characterized a Nb against
epidermal growth factor receptor (EGFR) and showed
in vitro inhibition of EGF binding to EGFR and in vivo
therapeutic effect on tumor in a mouse model (39).
Behdani et al have shown that the VEGFR2-specific Nb
can recognize antigen on the HUVEC cell surface and
can inhibit in vitro endothelial tube formation and could
be considered as a cancer therapy agent (36).
Although many VHH phage libraries have been
constructed so far, based on our surveys, no VHH
against TIM-3 has been reported in literature or in
industry. In the current study, we successfully prepared
a novel anti-human TIM-3 (CD366) Nb from a camel
immune library using phage display method. We
showed its high binding capacity to TIM-3 comparable
with a commercial antibody. Interestingly, this Nb
showed a high anti-proliferation effect on HL-60, an
AML cell line, which was comparable with, even more
than, the inhibitory effect of a standard anti-TIM-3
antibody.
Novel nanobody prepared against TIM-3 Homayouni et al
Iran J Basic Med Sci, Vol. 19, No. 11, Nov 2016
1207
Conclusion
Altogether, we have been described the successful
generation of an anti-TIM-3-specific Nb from an
immune camel library, followed by soluble expression
of VHH protein and its binding capacity to TIM-3
(CD366). We also showed its high ability to block
Gal9/TIM-3 stimulated proliferation of a leukemic cell
line in vitro.
Acknowledgement
This work was financially supported by Iran
National Science Foundation (Grant No. 92028659) and
Isfahan University of Medical Sciences (Grant No.
392532).
Conflict of interest
The authors declare that there is no conflict of
interests regarding the publication of this paper.
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