Keywords monoclonal antibodies - TIGIT - PVR - tumor immunotherapy - antibody-dependent cell-mediated
cytotoxicity
Introduction
Immunotherapies have become a pillar of cancer therapy, and the treatments involve
use of immune checkpoint inhibitors (ICIs), chimeric antigen receptor T cell therapy,
and cancer vaccine.[1 ]
[2 ] Of particular interest is the clinical development of PD-1/PD-L1 antibodies that
have become a monument in cancer treatment with more indications for the use of them
being increasingly approved in recent years.[3 ] However, only a few patients showed durable clinical efficacy from ICIs, because
the inhibitory factors, such as cytokine IL-10 (interleukin-10), hypoxia, tumor metabolites,
and other immune checkpoints, in the tumor microenvironment (TME) promote the dysfunction
of tumor-infiltrating lymphocytes (TILs).[4 ] Therefore, combination of different immunotherapies will offer a promising revenue
and improve clinical outcomes of cancer patients in the future.[5 ]
The T cell Ig and ITIM domain (TIGIT) is an emerging immune checkpoint. Its structure
contains an extracellular immunoglobulin (Ig) variable domain, a type 1 transmembrane
domain and a cytoplasmic tail that involves an immunoreceptor tyrosine-based inhibitory
motif (ITIM) and an Ig tail-tyrosine-like motif.[6 ]
[7 ] The TIGIT ligand is mainly expressed on the surface of NK (natural killer cell),
T cells, and can bind to its receptor—poliovirus receptor (PVR, CD155)—a member of
the nectin-like family of adhesion molecules, which is highly expressed by many tumor
cells and associated with tumor progression.[8 ] The interaction between TIGIT and CD155 suppresses the immune responses of lymphocytes
through increased secretion of IL-10 and transmits inhibitory signals in the cytoplasmic
compartment.[9 ]
[10 ] The expression of TIGIT on TILs is upregulated in various types of malignancies,
and positively correlated with the expression of other inhibitory receptors, such
as PD-1. Thus, blockage of TIGIT and PD-1/PD-L1 may restore the function of TILs and
enhance the secretion of the antitumor interferon-γ (IFN-γ).[11 ]
[12 ]
In addition, the immune checkpoint CD96 can also bind to CD155 receptor and generate
immunosuppressive signals in NK or T cells. Therefore, blocking CD155 receptor may
simultaneously inhibit the binding of TIGIT and CD96 to CD155.[13 ] Although there have been many studies and clinical trials on anti-TIGIT monoclonal
antibodies (mAbs), few reports on the therapeutic effect of anti-CD155mAb are available,
thus targeting CD155 may have a great potential in anticancer therapy.[14 ]
Based on a previous research, expression plasmids for the light and heavy chains of
anti-TIGIT and anti-CD155mAbs were first constructed in this work.[15 ] Then a large amount of anti-TIGIT and CD155 antibodies were expressed transiently
via the mammalian transient expression system. The products were purified through
affinity purification and high-purity proteins were obtained for biological evaluations.[16 ] We also established a simple TME model to assess the content of TIGIT receptors
on the surface of T cells after co-culture with tumor cells. Then, antibody-dependent
cell-mediated cytotoxicity (ADCC), antibody binding affinity, and the antitumor efficacy
of the antibodies were evaluated in vitro .[17 ] Preliminary results showed that CD155mAb has a more potent in vitro antitumor activity than TIGIT mAb. We also demonstrated that the in vitro antitumor effect of the combination of TIGIT mAb and CD155mAb was comparable to the
combination of TIGIT and PD-L1.
Materials and Methods
Reagents and Antibodies
The reagents and antibodies used in the study included: 25kDa linear polyethyleneimine
(PEI; Polyscience, United States); PrimeStar mix PCR polymerase, Hind III restriction enzyme, Nhe I restriction enzyme (TAKARA Bio, Japan); Ficoll-Paque (GE Healthcare, United States);
CD155 and TIGIT recombinant protein (Sino Biological Inc., Beijing, China); Donkey
Anti-Human IgG (H+L) Secondary Antibody (cat# 709–035–149; The Jackson Laboratory,
United States); goat anti-human IgG (H+L) secondary antibody, FITC (cat# 31529; Thermo
Fisher Scientific, United States); anti-TIGIT FITC (cat# 53–9500–42; eBioscience,
United States); CFDA (cat# 40715ES25; Yeasen Biotechnology (Shanghai) Co., Ltd., China),
SE Cell Proliferation Tracer Fluorescent Probe and propidium iodide (PI; cat#40710ES03;
Yeasen Biotechnology (Shanghai) Co., Ltd., China); anti-CD3, anti-CD28, anti-CD4,
and anti-CD8 PE antibody (Sino Biological); PD-L1 (Tecentriq) antibodies were expressed
by 293F cell and preserved in our laboratory. Analytical reagents such as sodium chloride,
citric acid monohydrate, and disodium hydrogen were purchased from Sinopharm Chemical
Reagent Co., Ltd. ClonExpress MultiS One Step Cloning Kit (cat# C113–01) was obtained
from Vazyme, China; AxyPrep DNA Gel Extraction Kit was purchased from Axygen, United
States; Endotoxin-Free Plasmids Extraction Kit was obtained from Omega, United States;
and human IFN-γ Enzyme-Linked Immunosorbent Assay (ELISA) Kit was obtained from Sino
Biological, China. Freestyle 293, F12K, RPMI1640, and DMEM medium, as well as trypsin
and fetal bovine serum (FBS) were purchased from Gibco (United States). Enhanced Chemiluminescent
(ECL; cat# P10300) was purchased from New Cell & Molecular Biotech (Suzhou) Co., Ltd.
Cells
U251MG and A549 cells were purchased from the Chinese Type Culture Collection. HEK293F
and huTIGIT-293T cells were maintained in our laboratory. All cell lines were cultured
at 37°C with 5% CO2 in the incubator. Fresh human peripheral mononuclear cells (PBMCs) were separated
from healthy donors in Changhai Hospital of Shanghai; Escherichia coli DH5α was purchased from Suzhou NCM Biotech.
Plasmid Construction
The light and heavy chain variable region sequences of anti-TIGIT and anti-CD155mAb
are referred from two patents respectively.[18 ]
[19 ] When designing the antibody expression sequence, 15bp of homologous sequences were
added to the front end of the enzyme cutting site Hind III and the end of NheI respectively, and then delivered to Shanghai Sangon Biotech to synthesize the sequence.
The synthesized light and heavy chain DNA vectors were cut from the original vector
by restriction endonuclease, and then the target fragment was obtained by Gel Extraction
Kit. The ADCC function was attenuated by using mutations at L234A, L235A, and P329G
(LALA-PG) in the Fc region, then the heavy-chain variable regions of the two antibodies
were linked with the mutated Fc domains by using homologous recombination.
Protein Expression and Purification
The six expression plasmids were extracted by an endo-free plasmid extraction kit,
and all plasmids were sterilized by 0.22 μm filter. The light and heavy chain plasmids
were mixed at a mass ratio of 1:1, then added PEI agent at a mass ratio of DNA:PEI=1:4,
and incubated at room temperature for 15minutes. Lastly, the DNA blend was added to
the HEK293F cell suspension and incubated in a 37°C shake incubator for 5 days. Then,
cell supernatant was collected by centrifuge (4,000g , 20minutes), and filtered through a 0.45 μm filter. We connected the Protein A column
on the AKTA Avant and equilibrated five column volumes with binding buffer, then loaded
the sample via the Avant. After loading the sample, the impurities were washed with
pH 5.0 citrate buffer and antibody was eluted with pH 2.7 citrate buffer and dialyzed
overnight to remove the eluate buffer. The above-mentioned buffer formula is shown
in [Table 1 ].
Table 1
Buffers
Buffer
Formula
0.1 mol/L citric acid buffer,
adjust pH to 5.0 and 2.7
21.014 g citric acid monohydrate+1,000mL H2 O
200mmol/L NaH2 PO4
23.996 g NaH2 PO4 +1,000mL H2 O
200mmol/L Na2 HPO4
28.392 g Na2 HPO4 +1,000mL H2 O
Binding buffer, adjust pH to 7.4
200mmol/L NaH2 PO4 (28mL), 200mmol/L Na2 HPO4 (72mL), 8.766 g NaCl+900mL H2 O
Antibodies' Characterization
The antibodies were separated by SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel
electrophoresis), and the molecular weight of the antibodies was analyzed by Coomassie
brilliant blue and Western blot. Briefly, the antibodies and protein markers were
added to gel holes of SDS-PAGE to run at a constant voltage of 90 V for 100 minutes.
The gel was cut, stained with Coomassie brilliant blue, and then washed with destaining
solution. Proteins on SDS-PAGE gel were transferred onto a PVDF membrane, and incubated
with Donkey Anti-Human IgG (H + L) Secondary Antibody for 1 hour. Visualization was
performed by chemiluminescence of membranes using ECL reagents. Purity of the two
antibodies was determined by the size exclusion chromatography (SEC-HPLC) method.
Affinity Assay by ELISA Method
The ELISA method was used to assess the affinity of the antibodies to antigens. Briefly,
TIGIT and CD155 recombinant proteins were coated overnight at a concentration of 25ng/mL,
and the two mAbs were serially diluted and added to the coated antigen. After 1hour
of incubation, added peroxidase-labeled donkey anti-human IgG (H+L) antibody to incubate
and conducted a color reaction after the end of the incubation, and placed it in a
microplate reader to measure the absorbance at OD450nm . The EC50 value was determined as the concentrations of the antibody used for half-maximal
of OD450nm . The K
d values were determined as equilibrium dissociation constant of antigen/antibody complexes
in solution.
Affinity Assay by Flow Cytometry
A549 and U251 cells were cultured in DMEM containing 10% FBS, while CHO-s cells were
cultured in F12K medium containing 10% FBS. The A549 and U251 cells were digested
with trypsin, and adjusted the cell count to 105 cells per tube, and anti-CD155 or anti-TIGIT mAb was added and incubated on ice for
30minutes. After the incubation, the cells were incubated with goat anti-human IgG
(H+L)-FITC antibody for another 30minutes, and the samples were loaded and analyzed
by a flow cytometer.
Tumor Cell and PBMC Co-culture Experiment
A549 and U251 cells were digested by trypsin. After centrifugation (300g , 3minutes), the supernatant was removed and the cells were resuspended (density 1.0×106 cells/mL). The cells were then seeded into a 96-well plate at 104 cells per well and cultured for 12hours. After the cells have completely adhered
to the wall, PBMCs were added at an effect-to-target ratio of 1:5. Blank wells contained
PBMCs without target cells and positive controls were PBMCs activated by CD3/CD28
antibodies (CD3: 5μg/mL, CD28: 2μg/mL). After 24hours of culture, the suspended PBMCs
were gently collected. Anti-CD4 (PE), anti-CD8 (PE), and anti-TIGIT (FITC) antibodies
were added to collected PBMCs, then were incubated at 4°C for 30minutes. After the
incubation, the cells were washed three times with 2% FBS-PBS buffer and evaluated
by flow cytometry.
Antibody-Mediated Cytotoxicity against A549 and U251 Cells
CHO-s, A549 and U251 cells were digested with trypsin, and the cell count was adjusted
to 106 cells/mL. CFDA was added to the cell suspension (the final concentration of CFDA
was 5 μmol/L) and placed in a 37°C cell incubator for 30minutes. After the incubation,
the cells were washed three times with PBS, and then added to a 96-well culture dish
at 104 cells per well. Human PBMCs are isolated by Ficoll density gradient centrifugation,
and cultured with 10% FBS RPMI-1640. The next day, PBMCs were added to 96-well cell
culture dishes at a ratio of 10:1 target cells and added different antibodies or different
concentration to each group. After co-cultivation for 24 or 48hours, the cell supernatant
was collected by centrifugation (300g , 3minutes), and the adherent cells were detached and resuspended in PBS. Finally,
PI dye (final concentration 0.3mmol/L) was added to each sample and incubated for
15minutes at room temperature for flow cytometry analysis. IFN-γ content in cell culture
supernatant was determined by IFN-γ ELISA kit according to manufacturer's instructions.
The cell killing rate was calculated according to [Eqn. (1) ] and cell viability (%)=CFSE + PI− single positive cells.
Statistical Analysis
Data were presented as the mean of at least two replicate samples and standard errors.
Student's t -test was used to compare between groups with p <0.05 being considered a statistically significant difference.
Results
Construction of Expression Plasmids for Anti-TIGIT and Anti-CD155mAbs
The expression plasmid was pcDNA3.4 ([Fig. 1A ]), and the plasmids to be constructed in the study included the heavy chain, the
light chain, and the LALA-P329G mutant heavy chain of two antibodies ([Fig. 1B ]). The heavy chain and light chain of two antibodies were obtained by enzyme digestion
of synthesized sequence fragments, and the size of the heavy chain was approximately
1,500bp, and the light chain was approximately 700bp ([Fig. 1C ]). The target fragments were purified by gel extraction. The fragments and the vector
were linked through the homologous recombination. Finally, bacterial transformation
was performed and positive clones were selected. The expression vectors were extracted
and sterilized through a 0.22 μm filter.
Fig. 1 Construction of expression vector. (A) pcDNA3.4 expression vector map. (B) Schematic diagram of different expression vectors. (C) Agarose gel images of light and heavy chains of TIGIT and CD155 antibody after digestion.
TIGIT, T cell Ig and ITIM domain.
Antibody Purification and SEC-HPLC Analysis
Protein A affinity chromatography was used for antibody purification. Results of nonreducing
([Fig. 2Aa ]) and reducing ([Fig. 2Ab ]) SDS-PAGE showed that the antibody band was approximately 180kDa. The sizes of heavy
chain and light chain were approximately 55 and 25kDa, respectively. These bands of
antibodies indicate that they were close to the theoretical molecular weight of the
IgG-like antibody with high purity. [Fig. 2Ac ] shows that the IgG-like antibodies we expressed were specifically bound by the anti-human
IgG (H+L) secondary antibody. We measured the concentration of two purified mAbs using
a BCA kit that showed CD155mAb being 50mg/L, and TIGIT mAb 20mg/L.
Fig. 2 Analysis of the molecular weight and purity of purified antibodies. (A) Analysis of antibody molecular weight by (a ) nonreducing SDS-PAGE; (b ) reducing SDS-PAGE; and (c ) Western blot. (B) Absorbance chromatogram of CD155mAb at UV 280nm by SEC-HPLC. (C) Absorbance chromatogram of TIGIT mAb at UV 280nm by SEC-HPLC. mAb, monoclonal antibody;
TIGIT, T cell Ig and ITIM domain.
SEC-HPLC analysis showed a high purity of the two mAbs ([Fig. 2B, C ]). We determined the purity by calculating the ratio of the main peak areas of the
two antibodies with CD155mAb and TIGIT mAb at 91.54 and 95.6%, respectively. The results
of SEC-HPLC were consistent with the results of SDS-PAGE, and the purity of purified
antibodies was greater than 90%. Therefore, the expression system of transient transfection
of 293F cells readily produced mAb with high yield and purity.
Antibody Binding of TIGIT and CD155 Antigens
Flow cytometry was used to investigate CD155 expression on U251 and A549 cells according
to reports.[20 ]
[21 ] We used anti-CD155mAb as a primary antibody to bind the CD155 receptor on the surface
of U251 and A549 cells. Anti-human (H+L) FITC secondary antibody was added to capture
primary antibodies. The data showed that both fluorescence intensities of U251 and
A549 were shifted to the right, but the control CHO-s cells showed no shift ([Fig. 3 ]). Therefore, we chose U251 and A549 as model cell lines in biological experiments.
Fig. 3 Flow cytometry analysis showing CD155 expression on the surface of U251 and A549
cells.
Results from ELISA assay showed that both anti-TIGIT and anti-CD155 antibodies bound
TIGIT and CD155 recombinant protein antigens with high affinity ([Fig. 4A ]). The EC50 of TIGIT mAb was 0.00714μg/mL, and the EC50 of CD155mAb was 0.01111μg/mL ([Table 2 ]). The affinity of TIGIT mAb appeared to be higher than that of CD155mAb; therefore,
subsequent experiments required adjustment of antibody concentration. Flow cytometry
showed that the anti-CD155mAb specifically targeted U251 cells and anti-TIGIT mAb
bound to TIGIT overexpressing 293T cells, indicating theses mAbs bound cellular receptors
with high affinity. Flow cytometry also showed ([Fig. 4B ]) that the EC50 of TIGIT mAb was 0.09716μg/mL, and the EC50 of CD155mAb was 0.1377μg/mL ([Table 3 ]). The difference between the cytometry result and the ELISA assay suggests that
the counts of cell surface receptors were not homogeneous.
Fig. 4 The affinity of two antibodies to antigens and receptors detected by (A) ELISA and (B) flow cytometry. In flow cytometry, the affinity of TIGIT mAb for TIGIT receptor was
detected on the surface of TIGIT-overexpressing 293T cells, while CD155mAb for CD155
receptor was detected on the surface of U251 cells. ELISA, enzyme-linked immunosorbent
assay; mAb, monoclonal antibody; TIGIT, T cell Ig and ITIM domain.
Table 2
EC50 and K
d values for different antibodies to antigens measured by ELISA
TIGIT
TIGIT mAb
CD155
CD155mAb
EC50 (µg/mL)
0.00714
EC50 (µg/mL)
0.0111
K
d (µg/mL)
0.00717
K
d (µg/mL)
0.0113
Abbreviations: ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody;
TIGIT, T cell Ig and ITIM domain.
Table 3
EC50 and K
d values for different antibodies to antigens measured by flow cytometry
TIGIT
TIGIT mAb
CD155
CD155mAb
EC50 (µg/mL)
0.09716
EC50 (µg/mL)
0.1377
K
d (µg/mL)
0.09280
K
d (µg/mL)
0.1377
Abbreviations: mAb, monoclonal antibody; TIGIT, T cell Ig and ITIM domain.
Upregulation of TIGIT on T Cells after Co-culture with Tumor Cells
A previous study demonstrated that the expression of TIGIT on the T cell surface was
effectively enhanced after stimulating PBMC with CD3/CD28 antibodies.[6 ] Treating tumors with anti-TIGIT restored the function of immune cells against tumor
cells.[22 ] We used activation antibodies (CD3/CD28) to treat PBMC and tested the expression
of TIGIT on T cells. Our data suggest that after activation, the proportion of CD4+ TIGIT+ T cells increased from 2.71 to 4.09%, and CD8+ TIGIT+ T cells also increased from 2.39 to 10.1% ([Fig. 5 ]). We then established a simple model by co-culturing A549 and U251 cells with PBMCs
and using flow cytometry to detect TIGIT expression on immune cells. We collected
PBMCs incubated for 24hours from a 96-well plate, then used CD3 antibody to distinguish
T cell populations, while PE-labeled anti-CD4 and CD8 antibodies were used to mark
T cell subsets. FITC-labeled anti-TIGIT antibody was finally used to verify the expression
level of TIGIT antibody.
Fig. 5 Flow cytometry analysis showing surface TIGIT expression after CD3/CD28 antibody
activation of PBMC. TIGIT, T cell Ig and ITIM domain.
After co-culture of PBMC with A549 and U251 cells, the TIGIT receptor on T cells was
significantly upregulated and the proportion of CD4+ TIGIT+ T cells increased from 2.68 to 18.8 and 26.8%, respectively, while CD8+ TIGIT+ T cells also increased from 0.98 to 25.7 and 18.5% ([Fig. 6 ]). The upregulation of TIGIT in this assay was higher than that of T cells activated
with CD3/CD28, which might be due to the fact that activation with CD3/CD28 required
a longer incubation time.[6 ] The inhibitory effect of tumor cells on T cells was related to the interaction between
TIGIT and CD155 to a certain extent, and this part of the results provided a theoretical
basis for the subsequent anti-TIGIT antibody-mediated antitumor experiments.
Fig. 6 Flow cytometry diagram of TIGIT expression on the surface of PBMC, A549, and U251
cells after co-culture for 24hours. TIGIT, T cell Ig and ITIM domain.
Antibody-Mediated Antitumor Effects
To directly measure tumor killing by lymphocytes, we stained tumor cells with CFSE
before co-culturing with PBMCs.[12 ] Tumor cells were divided into separate populations by CFSE staining and tumor killing
was revealed by PI staining of dead cells. As shown in [Fig. 7 ], anti-TIGIT and anti-CD155mAbs significantly enhanced the percentage of dead cells
(CFSE+ PI+ positive cells) compared to the control samples (PBS+PMBC). We then used cell viability
index (cell viability [%]=CFSE+ PI− single positive cells) to evaluate the killing effect of the antibody, because the
results using the CHO-s cell line showed that the antibody did not trigger PBMC to
produce killing effect on nontumor cell lines that did not express CD155 ([Fig. 8 ]). Thus, the cell viability rate was used to compare the results more clearly. As
shown in [Fig. 8 ], the target cell viability of the anti-CD155mAb group at different concentrations
was lower than that of the anti-TIGIT mAb group, and the two antibodies showed a killing
effect at a low dose of 2.5μg/mL. Therefore, we believe that blocking the combination
of TIGIT and CD155 may improve the PBMC killing effect on the CD155 high-expressing
tumor cell line.
Fig. 7 Flow cytometry results of lymphocyte killing of U251 tumor cells mediated by different
antibody combinations.
Fig. 8 The effect of different concentrations of TIGIT and CD155mAb on the viability of
U251, A549, and CHO-s cells in the presence of PBMC. mAb, monoclonal antibody; TIGIT,
T cell Ig and ITIM domain.
In addition, ADCC-enhancing antibodies were able to promote lymphocytes for killing
tumor cells, while ADCC-impairing antibodies (Fc silence) only slightly enhanced killing
of target cells ([Fig. 9A ]). The U251 cell line was more sensitive to PBMC than the A549 cell line. Therefore,
we confirmed that blockade of TIGIT and CD155 targets increased the antitumor effect
and the efficacy of CD155mAb was superior to that of TIGIT mAb. Also, the combination
of TIGIT and CD155mAbs showed a potential anticancer effect, but the combination therapy
did not show a difference from anti-CD155mAb in the A549 cell line. We further validated
the combination therapy of TIGIT and PD-L1mAb. Since anti-PD-L1 did not possess ADCC
function, we used CD155mAb without ADCC function as a control. The results indicate
that combination therapy of TIGIT+CD155mAb generated the best antitumor activity on
the U251 cell line ([Fig. 9B ]), while it did not show better effect than TIGIT+PD-L1 combination on A549 cells.
In addition, the killing rate of PD-L1mAb was weaker than that of CD155mAb, which
may be due to the different expression levels of PD-L1 and CD155 on cells. Therefore,
more models and experiments are required to determine whether the antitumor activity
of CD155mAb is more potent than PD-L1mAb.
Fig. 9 The effect of TIGIT and/or CD155 on killing and IFN-γ production of U251 and A549
cell lines. Statistics of the killing rate of U251 and A549 tumor cells by (A ) antibodies with or without ADCC effect and (B ) different antibodies. All data shown are representative of independent experiments
with at least two different PBMC donors replicated over three times. *p <0.05, **p <0.005, ***p <0.001. (C ) ELISA results of IFN-γ release in A549 and U251 cell culture supernatants. Experiments
were repeated three times. *p <0.05, **p <0.005. ELISA, enzyme-linked immunosorbent assay; IFN-γ, interferon-γ; TIGIT, T cell
Ig and ITIM domain.
ELISA results showed that TIGIT mAb with ADCC function promoted IFN-γ release, and
the effect was similar to CD155mAb without ADCC function ([Fig. 9C ]). However, PD-L1mAb without ADCC did not stimulate the release of a high level of
IFN-γ compared with other groups. Overall, combined treatment of TIGIT+PD-L1mAb and
TIGIT+CD155mAb promoted the release of a high level of IFN-γ to exert a more potent
antitumor effect ([Fig. 9C ]). The result was similar to that obtained in killing experiments. These findings
support that TIGIT and/or CD155 blockade increased cytokine production by lymphocytes
with enhanced cytotoxicity to tumor cells. The reason for the ability of CD155mAb
without ADCC function to promote a high level of IFN-γ release is unclear. We speculate
that CD155mAb may block another immunosuppressive checkpoint CD96.
Discussion
How to extend the clinical benefits to the majority of patients with tumors and explore
appropriate combination antitumor immunotherapies have been the key to this field.[5 ]
[11 ] As an emerging immune checkpoint, TIGIT has quickly entered the clinical trial;
the combination therapy targeting the TIGIT-CD155 axis will become a novel direction
of immunotherapy.[23 ]
There are thousands of mAbs in preclinical to early clinical development worldwide,
to accelerate the progress required for development of efficient methods to express
high yield and high purity mAbs. In our study, we expressed antibodies by transient
transfection of mammalian cells, which can not only raise the protein yield, but also
produce high-purity antibodies.[16 ] However, the transfection steps and purification conditions in the experiment need
to be optimized, such as the ratio of plasmid to PEI during transfection and the use
of different pH eluents to remove less pure proteins during the purification period.
In the SEC-HPLC analysis of the two expressed antibodies, we found that the purity
and yield were inconsistent, which may be due to different antibody variable region
sequences. The higher yield of CD155mAb compared to TIGIT mAb may be another reason
for the lower purity of CD155mAb. A higher expression efficiency may also generate
more impurities.
The binding activity of the antigen–antibody was the key to the efficacy of mAbs.
We have demonstrated that the expressed mAbs can bind to CD155 and TIGIT antigens
measured by ELISA and flow cytometry. But ELISA experiments were not the most accurate
method to determine the affinity constant values, so SPR (surface plasmon resonance)
assay was needed to evaluate affinity-related parameters of antibody–antigen binding.[24 ]
Both CD155 on tumor cells and TIGIT on lymphocytes were upregulated in TME, and their
interaction led to the depletion of immune cells.[25 ] Our study found that U251 and A549 cells have a high level of expression of CD155,
which impaired the killing function of immune cells due to the inhibitory signal between
TIGIT and CD155. We also demonstrated that TIGIT on the surface of T cells was significantly
upregulated when co-cultured with tumor cells, which may explain why PBMCs have limited
tumor-killing effect in the absence of anti-TIGIT and anti-CD155mAbs. Therefore, when
we added TIGIT and CD155mAb into PBMC, lymphocytes promoted the lysis of tumor cells
with the exception of the CHOs which did not express CD155 receptor. The effect of
CD155mAb is greater than that of TIGIT mAb, which led to our speculation that blocking
CD155 may inhibit simultaneously the two immune checkpoints of TIGIT and CD96. Consistent
with previous reports, we found that the ADCC effect contributed to the more potent
antitumor effect of TIGIT mAb that may be an important effect concerning this therapy.[26 ]
A study indicated that the efficacy of anti-TIGIT mAb and anti-PD-L1mAb alone in the
treatment of a mouse tumor model was similar to the results of our in vitro experiments.[22 ] We confirmed the conclusion that single treatment of TIGIT mAb was less effective,
and for the first time we observed that anti-CD155mAb has higher in vitro antitumor activity than anti-TIGIT mAb and anti-PD-L1mAb. Regardless of the ADCC
function of anti-CD155mAb, this mAb showed excellent in vitro antitumor effect, which was comparable to the combined use of TIGIT and PD-L1. Nevertheless,
the current research on the antitumor therapeutic effect of CD155mAb is not definite,
and limited studies showed that blocking CD155mAb decreases the invasion and migration
of tumor cells.[27 ] Another research indicates that both anti-TIGIT mAb and anti-CD155mAb were able
to enhance cytotoxicity mediated by bispecific antibodies targeting EGFR and CD3 with
similar therapeutic effects. However, the anti-TIGIT mAb and anti-CD155mAb used in
that study are commercial mAbs for proteomics, not for therapeutic purposes.[28 ] In addition, we evaluated the antitumor potential of various antibodies by measuring
the content of IFN-γ in cell culture supernatant with results similar to those of
the killing experiment. Surprisingly, CD155mAb without ADCC function still promoted
strong IFN-γ release, while the release amount caused by PD-L1mAb without ADCC function
was only stronger than that of the PBS group. We believe the reasons for this interesting
phenomenon are worth further exploration.
At present, the combination therapy of anti-TIGIT and anti-PD-L1mAb has entered the
clinical trial, but with only limited studies on the therapeutic effect of anti-CD155mAb.[29 ] Thus, our experiment indicates the potential antitumor effect of anti-CD155mAb.
Further animal experiments are required to examine whether the antibody may show functions
other than blocking the TIGIT-CD155 signal.
Conclusion
We have demonstrated that anti-CD155mAb and anti-TIGIT mAb stimulated T cells to release
cytotoxic cytokines to kill tumor cells. In vitro experiments showed that anti-CD155mAb was superior to the anti-TIGIT mAb in antitumor
efficacy. Combination of the antibodies demonstrates promising potential as a novel
cancer immunotherapy.
