Animal Biotechnology

ISSN: 1049-5398 (Print) 1532-2378 (Online) Journal homepage: http://www.tandfonline.com/loi/labt20

Generation and characterization of avian-derived

anti-human CD19 single chain fragment antibodies

Keng-Chang Tsai, Chen-Wei Chiang, Yan-Ni Lo, Fu-Ling Chang, Tsai-Yu Lin,
Chang-Yu Chang, Wang-Chuan Chen & Yu-Ching Lee

To cite this article: Keng-Chang Tsai, Chen-Wei Chiang, Yan-Ni Lo, Fu-Ling Chang, Tsai-Yu Lin,
Chang-Yu Chang, Wang-Chuan Chen & Yu-Ching Lee (2018): Generation and characterization
of avian-derived anti-human CD19 single chain fragment antibodies, Animal Biotechnology, DOI:
10.1080/10495398.2018.1486323

To link to this article: https://doi.org/10.1080/10495398.2018.1486323

Published online: 27 Sep 2018.

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ANIMAL BIOTECHNOLOGY
https://doi.org/10.1080/10495398.2018.1486323

ORIGINAL ARTICLE

Generation and characterization of avian-derived anti-human CD19 single

chain fragment antibodies



Keng-Chang Tsaia,b, , Chen-Wei Chiangc, , Yan-Ni Loc, Fu-Ling Changd, Tsai-Yu Linc, Chang-Yu Change,
Wang-Chuan Chenf,g and Yu-Ching Leec,d
a
National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan; bThe Ph.D. Program for Medical
Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan; cResearch Center of Cancer
Translational Medicine, Taipei Medical University, Taipei, Taiwan; dGraduate Institute of Cancer Molecular Biology and Drug Discovery,
College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan; eDepartment of Medical Technology, Jen-Teh
Junior College of Medicine, Nursing and Management, Miaoli, Taiwan; fThe School of Chinese Medicine for Post Baccalaureate, I-Shou
University, Kaohsiung, Taiwan; gDepartment of Chinese Medicine, E-Da Hospital, Kaohsiung, Taiwan

ABSTRACT KEYWORDS
The human cluster of differentiation 19 (CD19) is highly expressed in most leukemia, rendering Human cluster of
is a promising therapeutic target. In this study, we generated anti-CD19 single-chain variable differentiation 19; leukemia;
fragments (scFv) from immunized chickens by phage display technology. After constructing a single chain variable
fragment; phage display
scFv antibody library with 2.5  108 compositional diversity for panning, one representative technology; molecu-
scFv clone S2 which can specifically recognize to the CD19 protein was isolated and character- lar modeling
ized. The binding reactivity of the scFv S2 to the endogenous CD19 protein of the ARH-77
leukemia cancer cell was verified through flow cytometry and the binding affinity of scFv S2
is 6.9  108 M determined by the surface plasmon resonance system. Compared with the
chicken germline, hyper mutation in the complementarity-determining regions (CDRs) sug-
gested that scFv S2 could be generated through an antigen-driven humoral response. By
molecular modeling, the possible CDR configurations of scFv S2 were constructed rationally.
Furthermore, the characteristics of chicken antibodies of a protein database were investigated.
The findings in this study contribute to antibody development and engineering because they
reveal the geometric structures and properties of the CDRs in chicken antibodies.

Introduction class II molecules and signal transduction in vivo. The

The human cluster of differentiation 19 (CD19) anti-
gen, belonging to the immunoglobulin (Ig) superfam-
ily, is a transmembrane glycoprotein. It has no
significant homology to other known proteins and is
expressed from preB-cells until the terminal differenti-
ation of plasma cells as a reliable surface marker.1,2
Although whether CD19 is directly involved in B-cell
carcinogenesis is unclear, high expression levels of
CD19 have been found in approximately 80%–100%
of major B-cell malignancies such as acute lympho-
blastic leukemias (ALL), chronic lymphocytic leuke-
mias (CLL) and B-cell lymphomas.2,3 However, other
B-cell malignancies have relatively diminished CD19
levels.4,5 CD19 levels can also be used to differentiate
certain subtypes of lymphomas. Moreover, CD19 may
play a key role in regulating the expression of MHC
presented findings illustrate the pertinence of
researching CD19 because it may become an essential
therapeutic target for various autoimmune diseases.6
For clinical applications, CD19 monoclonal antibodies
have been developed, approved, and validated. The
United States Food and Drug Administration recently
approved Blinatumomab, which is a CD19/CD3-bispe-
cific T-cell engager directed against CD19 with unique
antitumor efficacy for treatment of B-cell ALL.7,8 In
addition to bispecific antibodies, the development of
chimeric antigen receptor technology with adoptively
transferred T cells expressing CD19-specific antibody
against B-cell malignancies is rapidly evolving and is
highly expected.9,10
The first recombinant antibody library was estab-
lished in 1989, which opened an efficient route

CONTACT Yu-Ching Lee ycl@tmu.edu.tw Research Center of Translational Medicine, Taipei Medical University, #250 Wu-Hsing Street, Taipei
110, Taiwan.
Supplemental data for this article can be accessed here.


These authors contributed equally to this work.
ß 2018 Taylor & Francis Group, LLC.
2 K.-C. TSAI ET AL.
through phage display technology for generating
monoclonal antibodies.11 Numerous antibodies gener-
ated through this novel technique are currently
being developed for target therapy.12,13 Traditional
approaches (such as hybridoma) for generating mono-
clonal antibodies are complicated and time consuming
and have numerous difficulties.14 Alternatively, the
phage display system is a safe and effective in vitro
procedure for enriching specific antibodies from com-
plicated phage antibody libraries.15 In general, small
mammals such as mouse and rabbit are often used
because of their convenient operation. However, it is
frequently to develop tolerance with highly conserved
proteins of mammals during immunization due to
phylogenetic relationship. This issue limits experimen-
tal options and hinders the progress of research. To
solve this problem, the chicken is a suitable host choice
because it can be used to induce stronger humoral
responses based on species differences. The chicken
has only a lambda light-chain isotype, compared with
two light-chain isotypes (kappa and lambda) in mam-
mals, facilitating the construction of an antibody
library for efficient binder screening.16 Moreover, sin-
gle-chain variable fragment (scFv) antibodies based on
chicken Ig frameworks often exhibit high expression
levels in simple bacterial cultures.17 Therefore, lots of
studies have examined the various applications of
chicken-derived scFv antibodies.18,19 Although the
humanization of chicken scFv antibodies is still
required for potential therapeutic use in the future, this
obstacle could be overcome through antibody
engineering.
We conducted this study to generate specific anti-
CD19 chicken-derived scFv antibodies, with a focus
on investigating the complementarity-determining
region (CDR) structure within the most acceptable
scaffold through molecule modeling. Moreover, the
characteristics of antibody sequences by the published
chicken antibody structure were explored. The results
from this study can provide an improved understand-
ing of the chicken scFv antibodies, the possible appli-
cations of which can be explored in future research.
Materials and methods
Chicken immunization
Female white leghorn (Gallus domesticus) chickens
were maintained carefully in the animal facility of the
Taipei Medical University. All of the animal experi-
ments followed ethical standards, with the protocols
having been reviewed and approved by the Animal
Use and Management Committee of Taipei Medical
University. The chicken was immunized with 10 lg
recombinant CD19 protein (Sino Biological Inc.,
China.) with an equal volume of Freund’s complete
adjuvant through intramuscular injection. Three add-
itional immunizations were carried out at intervals of 7
days. After final immunization, IgY antibodies in egg
yolks were purified and titrated through an enzyme-
linked immunosorbent assay (ELISA) to determine the
presence of a humoral immune response. The purifica-
tion of IgY from the egg yolks was in accordance with
a published protocol.20,21 The purified IgY antibodies
from each egg were dissolved in 5 mL of Tris-buffered
saline containing 0.05% sodium azide and stored
at –20  C.
Construction of chicken scFv library
and biopanning
A phage library displaying the scFv antibody was con-
structed according to published protocols with minor
modifications.16,19 Briefly, chicken spleens were har-
vested 7 days after the final immunization. After hom-
ogenization, total RNA was extracted and reverse
transcribed into the first-strand cDNA by using a
SuperScript RT kit (Invitrogen, USA). Chicken scFv
primers and PCR conditions were provided in the
supplementary files. After amplification using chicken-
specific primers, gene products of heavy-chain and
light-chain variable (VH and VL, respectively) regions
were subjected to a second round of polymerase chain
reaction (PCR) with a linker to form full-length scFv
molecules, which were cloned further into the
pComb3X vector.16 The recombinant DNA was trans-
formed into the Escherichia coli ER2738 strain through
electroporation, and the VCS-M13 (Stratagene, USA)
helper phage was then added to initiate recombinant
phage production. The total phage library was precipi-
tated with 4% polyethylglycol 8000 and 3% NaCl (w/v)
and resuspended in phosphate-buffered saline (PBS)
containing 1% bovine serum albumin (BSA) and stored
at 4  C.
For biopanning, 1011 recombinant phage particles
from the constructed scFv antibody library were
added to a microtiter plate precoated with a recom-
binant CD19 protein (1 lg/well) and incubated at
room temperature for 2 h. Next, unbound phage par-
ticles were removed and the well was washed with
PBST (PBS with 0.05% Tween 20) for 10 times.
Bound phage particles were eluted with 0.1 M HCl/gly-
cine (pH 2.2)/0.1% BSA elution buffer, neutralized
with 2 M Tris base buffer, and immediately used to
infect the E. coli ER2738 strain for recombinant phage

amplification. Phage particles were precipitated and
recovered as described previously and used for the
next round of panning. The panning procedure was
repeated four times. After panning, total library DNA
was purified by the EasyPrep Plasmid Extraction kit
(BioTools Inc., Taiwan) and transformed into E. coli
TOP 10F’ (Invitrogen, a nonsuppressor strain) for
scFv expression. The expressed scFv were further
purified with Ni2þ-charged sepharose according to the
manufacturer’s instructions (GE Biosciences, UK).
Western blotting and ELISA
To detect the binding reactivity of the selected scFv
antibodies, the recombinant CD19 protein was trans-
ferred onto nitrocellulose membranes (GE Biosciences)
after sodium dodecyl sulphate-polyacrylamide gel elec-
trophoresis. After being blocked with 5% skim milk,
the membranes were incubated with purified scFv anti-
bodies at room temperature for 1 h. Therefore, the
membranes were incubated and detected with goat
anti-chicken light-chain antibodies (Bethyl
Laboratories, Inc. USA) and developed with horserad-
ish peroxidase (HRP)-conjugated donkey anti-goat Ig
G (IgG) antibodies (Jackson ImmunoResearch
Laboratories, Inc., USA). A diaminobenzidine substrate
was added until the desired intensity was reached.
For ELISA, a microtiter plate was precoated with
0.5 mg/well of the recombinant CD19 protein at 4  C
overnight. After being blocked with 5% skim milk, the
poly-IgY or scFvs or scFv-expressing phages were
added to the wells in duplicate and incubated for 1 h
at room temperature. After washing with PBST, the
bound poly-IgY or scFvs were detected as described
previously and the scFv-expressing phages were
detected using HRP-conjugated mouse anti-M13
phage antibodies (GE Biosciences). Finally, the 3,5,5-
tetramethubezidine dihydrochloride substrate was
added to the wells for development. The reaction
was stopped by adding 1 N HCl, and absorbance was
measured at 450 nm.
Sequence analysis
To sequence the scFv clones, a primer ompseq (50 -
AAGACAGCTATCGCGATTGCAGTG-30 ) comple-
mentary to the outer membrane protein A (ompA)
signal sequence in front of the VL region was used.
The International ImMunoGeneTics information sys-
tem/V-QUEry and STandardization (http://imgt.cines.
fr/) was used to compile and analyze the sequence
data in accordance with the germline gene.
ANIMAL BIOTECHNOLOGY 3
Flow cytometry analysis
The ARH77 (CD19þ) leukemia cells were cultured in
Roswell Park Memorial Institute 1640 medium supple-
mented with 10% fetal bovine serum and 2 mmol/L
glutamine in vitro. A total of 2  106 cells were har-
vested and fixed with 2% paraformaldehyde. The
endogenous CD19 molecule expressed on the ARH77
leukemia cells was detected with purified chicken scFv
antibodies and following goat anti-chicken light chain
antibodies (Bethyl Laboratories, Inc.). The bound scFv
was visualized with fluorescein-isothiocyanate-conju-
gated donkey antigoat IgG antibodies (Jackson
ImmunoResearch Laboratories, Inc.). The results were
analyzed using a FACScan flow cytometer (Becton
Dickinso, USA).
Surface plasmon resonance
Binding analysis of scFv antibodies to CD19 was per-
formed using an OpenSPR instrument (Nicoya
Lifesciences, Canada). For scFv expression, a single
biotin was labeled on a unique peptide tag behind the
scFv by using the biotin ligase from E. coli.22 Briefly,
100 lL of the bioton-labeling scFv (100 lg/mL) in PBS
was provided as the target and immobilized on a strep-
tavidin sensor chip. In operation, the running buffer
was PBS, and a constant flow rate of 20 lL/min was
used. The CD19 molecule was dissolved in the running
buffer at concentrations of 1.25–10 lM for the experi-
ment. The sensor chip was regenerated using 10 mM
glycine-HCl (pH 2) at a flow rate of 150 lL/min after
each injection of scFv S2. Finally, the data were
recorded and analyzed through Trace Drawer software
(Ridgeview Instruments, Sweden) in accordance with
the manufacturer’s instructions. The kinetic parameters
of the interaction of scFv S2 with the CD19 protein
were investigated through TraceDrawer software
according to a 1:1 binding model.
Homology modeling of the scFv S2 antibody
To obtain a reliable structural model of scFv S2, three
crystallographic structural templates – namely a
chicken anticardiac Troponin I scFv (Protein Data
Bank [PDB] id: 4P48), a chicken antiprostate specific
antigen scFv (PDB id: 4P49), and a chicken antiptau
Fab (PDB id: 4GLR) – from the PDB were used for
building the scFv S2 model. Their sequence similar-
ities to S2 were 89.4%, 85.2% and 85.9%, respectively.
Therefore, 4P48 was used as the major structural tem-
plate with the highest sequence similarity to construct
the scFv S2 scaffold model through the Model
4 K.-C. TSAI ET AL.

Antibody Framework module from Discovery Studio
v. 2017 (BIOVIA Inc., USA). The lowest PDF total
energy (probability density function energy) was
employed to select a reliable scFv S2 model among
200 models.
Results
Characterization of humoral IgY response in
the chicken
The recombinant CD19 protein used for immuniza-
tion was analyzed through SDS-PAGE, revealing an
approximate molecular weight of 47 kDa due to glyco-
sylation (Fig. 1a, lane CD19). The purified IgY anti-
bodies from the egg yolks were tested for their
binding activity to CD19 immobilized on nitrocellu-
lose membranes or ELISA plate wells. Through
Western blotting, polyclonal anti-CD19 IgY antibodies
obtained after the fourth immunization substantially
bound to CD19. By contrast, the IgY antibodies
obtained from the pre-immunized chicken egg showed
no reaction (Fig. 1a). In the ELISA, the IgY antibodies
prepared from the egg yolks after the fourth immun-
ization specifically bound to CD19 but not to BSA,
even when titered at a high concentration (Fig. 1b).
The result suggests that a strong humoral antibody
response was elicited in the chicken host.
Construction of scFv antibody library
and biopanning
The immunized chicken was sacrificed and total RNA
was extracted from the spleen cells to construct the
antibody library. The amplification of full-length scFv
gene fragments was performed using two consecutive
PCR steps (Supplemental Fig. 1). After the M13 helper
phage was used for rescue, a high-quality phage dis-
playing scFv library was constructed; the library com-
plexity was determined to be 2  108. To evaluate the
ratio of clones with correct inserts in the constructed
library, 30 clones of the library were selected to con-
firm the presence of a 750 bp fragment containing
both heavy and light chain genes by using colony
PCR. The result revealed that 93% (28/30) of the
clones selected from the library had correct gene
inserts (data not shown). Next, we performed four
rounds of panning to eliminate unwanted clones and
to enrich specific binders against CD19. As shown in
Fig. 2a, eluted phage titer of the final round was over
one order higher than that of the first round of pan-
ning. The composition of the antibodies in the ampli-
fied library was varied after panning. Moreover, we
confirmed the binding reactivity against CD19 of the
amplified phage form in each round of panning
through phage ELISA (Fig. 2b). Notably, compared
with the original library, the binding signal increased
with the number of panning cycles.
Characterization of anti-CD19 chicken scFv clones
After panning, we randomly selected 14 clones for
single colony analysis. The expression levels of IPTG-
induced scFv clones were tested for binding activity to
the CD19 recombinant protein. These selected scFv
clones showed significant binding activity to CD19
but not to BSA (Fig. 3a). Through sequencing, we
determined the nucleotide sequences of the variable
regions of heavy and light chain genes of the 14 clones.

Figure 1. Humoral antibody response in chicken after immunization. (a) Recombinant CD19 protein was visualized using
Coomassie blue staining or transferred onto a nitrocellulose membrane, which was subsequently probed with polyclonal IgY puri-
fied from chicken egg before immunization and after forth immunization. Arrows indicate the recombinant CD19 molecular weight
at 47 kDa. (b) Humoral IgY responses in chicken egg after the fourth immunization analyzed through ELISA. A series of diluted IgY
antibodies were examined for their binding activity to CD19 (solid bars) or BSA (open bars).
 ANIMAL BIOTECHNOLOGY 5

The results revealed that 13 clones shared identical
heavy and light chain genes, except for clone S4.
Corresponding to the sequencing results, 13 identical
scFv clones were clearly detected through Western
blotting, showing consistent patterns (Fig. 3b).
Because the scFv linker was designed to seven residues
(GGSSRSS), a short linker caused the scFv expression
trend to be a dimer form (50 kDa), which was more
than the expected monomer form (25 kDa). One rep-
resentative scFv S2 was expressed and purified for fur-
ther experiments. The purified scFv S2 was analyzed
by SDS-PAGE to confirm the purity as well
(Supplemental Fig. 2).
We also examined the recognition of scFv S2 to
recombinant CD19. The results showed that scFv S2
could clearly recognize the CD19 protein at an

Figure 2. Evaluation of panning process. (a) Eluted phage numbers were monitored after each round of panning against CD19.
Wild-type M13 phage was used in the panning process as a negative control. (b) The amplified phage library after each round of
panning was used to confirm the binding reactivity by using phage ELISA. Ori denotes original phage library before the pan-
ning process.

Figure 3. Characterization of isolated anti-CD19 scFvs. (a) Binding activity of randomly selected scFvs expressed in the cell lysate
to CD19 was examined using ELISA. (b) Western blotting of selected scFv expression. Arrows indicate the scFv molecular weight of
monomer or dimer forms. (c) Recombinant CD19 protein indicated with arrows were visualized using Coomassie blue staining or
detected using a representative scFv S2 through Western blotting. (d) Binding activity of scFv S2 to endogenous CD19 expressed
on the ARH77 leukemia cell membrane was analyzed through flow cytometry. PC denotes commercial anti-CD19 polyclonal anti-
bodies used in the test as a positive control. NC denotes an irrelevant scFv without a binding signal.
6 K.-C. TSAI ET AL.
expected molecular weight (Fig. 3c). By using flow
cytometry to test the binding reactivity of scFv to
endogenous human CD19 intrinsically expressed on
the membrane of ARH77 leukemia cells, we observed
that scFv S2 showed significant binding signals to
ARH77 tumor cells; we noted no binding activity
when we applied an irreverent scFv (Fig. 3d). A sur-
face plasmon resonance (SPR) system was used for
the assay to measure the binding affinity of scFv S2 to
the CD19 molecules (Supplemental Fig. 3). The KD
value of scFv S2 was 6.9  108 M, and the affinity
constants are presented in the Table 1. By nature rep-
ertoire after immunization, the affinity at pM level of
isolated scFv is generally acceptable. The result is
similar to previous studies.23,24
ScFv gene sequence analysis
The nucleotide sequences of scFv S2 were aligned to
the germline gene sequences of chicken Ig. As shown
in Fig. 4, the overall mutation rates as compared with
the germline gene sequences were 18.3% and 23.1% in
VL and VH regions, respectively. The mutation rate
in the CDRs was higher than that in the framework
regions (FRs). Insertion mutations occurred in the
heavy chain CDR3.
Homology modeling of scFv S2 structure
The structure of CDR loops of the chicken antibody
scFv S2 was constructed using homology modeling
strategies. Based on three x-ray crystallography
chicken antibody structures that have been published,
4P48 (PDB code) is a chicken anticardiac Troponin I
scFv that is most similar to the scFv S2 framework.
As shown in Fig. 5a, the lengths of the CDR-L1 and
CDR-L2 loops of scFv 4P48 are greater than those of
Table 1. kon and koff rate constants of chicken scFv S2 target-
ing to CD19 protein.
Ligand Analyte (scFv) kon (102 M1 S1) koff (105 S1) KD (108 M)
CD19 S2 6.32 ± 0.00317 4.39 ± 0.00623 6.94 ± 0.0718
Figure 4. Sequence analysis of VL and VH genes of the scFv S2 antibody. VH and VL nucleotide sequences were determined and
translated into amino acid sequences to be aligned with the chicken germline gene. Sequence gaps were introduced to maximize
the alignment; these are indicated by blank spaces. Dots indicate the consensus sequences. FR and CDR boundaries are indicated
above the germline sequences.
scFv S2. The extra amino acids in the CDR-L1 loop
are one glycine, one arginine, and two tyrosines,
whereas the CDR-L3 loop containe one aspartic
amino acid. The lengths of the other CDR loops are
the same and the CDR-H1 sequence is identical to
scFv S2. Comparing the constructed structure of scFv
S2 with scFv 4P48 reveal a shorter CDR-L1 (Fig. 5b
and c), and the variability of CDR-L1 is similar to
that of CDR-H3 having high tolerance to display on
the chicken antibody.
Discussion
Different from the traditional hybridoma approach,
the phage display system provides a molecular bio-
logical method for rapidly and conveniently preparing
monoclonal antibodies. However, when the scFv gene
is assembled in a library, the combination of heavy
chains and light chains is random, which may affect
the binding activity of the displayed scFv because of
the unsuitable structure. Nevertheless, such problems
can be ameliorated through affinity maturation by
using antibody engineering to enhance antibody avid-
ity, which has been widely used in the development of
many clinical antibody drugs. In this study, we used a
short linker (7 aa) in the structure of scFv, which
caused the scFv expression to become a dimer form
(Fig. 3b) to improve the selection efficiency based on
the binding affinity of the panning process. The bind-
ing affinity of scFv S2 identified through the SPR sys-
tem was 69 pM. The structural change caused by the
linker contribute to enhance the binding ability of
scFv. In Fig. 3b, scFv clone S4 has a molecular weight
larger than other clones in dimer form. After sequenc-
ing, the length of VH and VL genes of scFv S4 was
confirmed to be general. The aggregation of scFv is
often occurring via the domain-swapping manner.
Different antibody sequence associated with the vari-
able domain interface is one of the factors leading to
aggregation or multimer expression.25 As scFv S2 and
S4 have been heated before SDS-PAGE analysis, the
 ANIMAL BIOTECHNOLOGY 7

aggregation of scFvs were dissociated to monomer
(Supplemental Fig. 4).
Compared with the result obtained from the first
panning process (using the original library as an input
phage), the eluted phage number obtained from the
fourth panning process increased by 40 times, demon-
strating the enrichment of the specific antibody clone
in the library. Moreover, the eluted phage number
from the second to the fourth panning showed an
obvious upward trend, also suggesting successful selec-
tion. Similar results have been reported in our previ-
ous studies and other studies.26,27 Based on the
differences of species, the chicken tends to have more
somatic mutations after immunization with proteins
of mammalians. Notably, the somatic mutation of
scFv clones is mainly from amino acid insertion (Fig.
4) compared with the germline of chickens. This
means that somatic mutation causes high variability.
The greater length of CDR3-H3 is the basis for the
variable CDR, which has the ability to enter deeper
antigenic epitope regions.
To understand the differences in sequence between
the isolated chicken antibody (scFv S2) and human
antibodies, we conducted a preference analysis using
three published chicken antibody structures (4P48,
4P49 and 4GLR) and 10 representative human anti-
body structures (3G6A, 3NH7, 4QHU, 4QCI, 4OD2,
4QF1, 8FAB, 4HPY, 5BV7 and 1W72) from the PDB,
which all have high similarity to the framework
sequence of scFv S2 (Fig. 6a and b). The most signifi-
cant difference between the chicken and human anti-
body sequences was determined to be in the variable
region of the light chain. The length of the CDR-L1
of chickens was noted to be highly variable compared
with human CDR-L1, which has a fixed and con-
served length. This may be explained by this feature
of chicken antibodies to be able to have more effective
abilities. Notably, the chicken framework was observed
to have one more amino acid of serine or alanine
than the human sequence has at the position of 45A
(based on the numbering in the international
ImMunoGeneTics information system). The difference

Figure 5. Homology modeling of scFv S2 structure. (a) Multiple sequence alignment of variable domains of the light and heavy
chains of 4G48, 4P49 and 4GLR with the scFv S2 antibody. The background of the residues is colored according to sequence simi-
larity. The deep color shows the conserved residues in all sequences. The color scheme is from deep to light, corresponding to
identity, highly conserved residues, and low-conserved residues, respectively. The residue backgrounds colored in white are not
similar. Residues of the CDRs are indicated by bold letters with arrow marks. (b) PDB id: 4P48 was used as the major structural
template with the highest sequence similarity to construct the (c) scFv S2 scaffold model.
8 K.-C. TSAI ET AL.

Figure 6. Sequence preference analysis of published chicken and human antibody with scFv S2. Multiple sequence alignment of
variable domains of the light chains (a) and the heavy chains (b) of chicken antibodies (PDB id: 4G48, 4P49 and 4GLR) and the
representative nine human antibodies (PDB id: 3G6A, 3NH7, 4QHU, 4QCI, 40D2, 4QF1, 8FAB, 4HPY and 1W72) with the scFv S2
chicken antibody. All amino acids of sequences are renumbered using IMGT schemes. The background of the residues is colored
according to sequence similarity. The deep color shows the conserved residues in all sequences. The color scheme is from deep to
light, corresponding to identity, highly conserved residues, and low-conserved residues, respectively. The residue backgrounds col-
ored in white are not similar. Residues of the CDRs are indicated by bold letters with arrow marks.

in the position of chicken antibodies may be related
to the complexity tolerance of CDR-L1. The chicken
and human characteristics of the VH region were
determined to be similar, with all of them exhibiting a
high degree of CDR-H3 diversity. Therefore, chicken
antibodies can induce effective somatic mutations on
CDRL1 and CDRH3. Overall, this information affords
a clearer understanding of the characteristics of
chicken antibodies and can thus assist us by this way
to develop antibody drugs in the future. Such an
immune strategy can be used to induce highly specific
antibodies, and through antibody engineering, can

improve and develop the possibility of clinical
applications.
Disclosure statement
No potential conflict of interest was reported by
the authors.
Funding
This work was financially supported by the “TMU Research
Center of Cancer Translational Medicine” from The
Featured Areas Research Center Program within the frame-
work of the Higher Education Sprout Project by the
Ministry of Education (MOE) in Taiwan; Ministry of
Science and Technology in Taiwan under Grant [MOST
105-2628-B-038-007-MY3].
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