Use of Oncolytic Vaccinia Virus Armed with Certain Cytokines

(IL-7, IL-12, IL-15, IL-21) in Combination with Checkpoint

Inhibitors to Treat Non-Small Cell Lung Cancer

Yuqian Xu

1,*

, Yuantong Li

, Ziyang Yin

, Zihao Jin

, Wendao Li

and Zhaoyang Che

School of Pharmaceutical Sciences, Tsinghua University, Beijing, 100084, China

Department of Biology, Brandeis University, Waltham, MA, U.S.A.

Concordia International School Shanghai, Shanghai, 201206, China

Western International School Shanghai, Shanghai, 201206, China

Shenzhen Foreign Languages School, Shenzhen, 518083, China

Keywords:

Oncolytic Virus, Checkpoint Inhibitors (Cpis), Cytokines, Non-Small Cell Lung Cancer.

Abstract:

A variety of oncolytic viruses (OVs) have been reported in treating different cancers and several have entered

clinical trials. This study proposes the combination of cytokines IL-7, IL-12, IL-15, IL-21, and checkpoint

inhibitors (CPIs) co-expressed on an oncolytic vaccinia virus JX-594 to treat non-small cell lung cancer

(NSCLC) with the aim of tumor regression. The effectiveness of the modified JX-594 vaccinia virus is

measured both in H460 cell lines and in LSL-KrasG12D mouse model, including the production of cytokines

and CPI, cytotoxicity, tumor growth, overall survival and tumor-infiltrating lymphocytes (TILs). The

increased overall survival rate and tumor size regression in mouse model are predicted.

1 INTRODUCTION

1.1 General Overview to Oncolytic

Viruses

In 1991, a genetically engineered herpes simplex

virus-1 (HSV-I) was found to be a potential therapy

in malignant glioma (Martuza, 1991). Since then,

increasing attention was paid to the role of virus in

treating cancer. Nowadays, oncolytic virus (OV) is a

promising cancer immunotherapy. OV could kill

cancers in two main ways: direct cell lysis as well as

increased anti-tumor immune response (Abd-Aziz N,

2021). OVs are genetically engineered to selectively

infect cancer cells via some receptor-mediated

pathways, therefore minimizing potential tissue

damage (Ferguson, 2012). OVs could also stimulate

anti-tumor immune responses, such as presentation of

TSAs (tumor specific antigens) and TAAs (tumor-

associated antigens) to APCs to activate effector T

cells (Ferguson, 2012). As the knowledge of the

therapy advanced over years, OVs are genetically

modified in diverse ways to enhance antitumor effect.

Corresponding author.

These armed OVs could deliver cytokines, CPIs,

tumor suppressors, etc (Marintcheva, 2018). Besides,

a wider variety of OVs were tested clinically,

including adenovirus, reovirus, measles, HSV-I,

vaccinia virus, and Newcastle disease virus

(Fukuhara, 2016).

Despite the extraordinary advantages, current

oncolytic virus treatments face several challenges,

including physical barriers, antiviral immunity, and

immunosuppressive tumor microenvironment

(TME). Multiple strategies that enhance OV delivery,

infiltration, and oncolysis have been developed to

increase the efficacy of OV therapy. Besides,

recombinant OVs might cause some unexpected

toxicities so safety analysis must be considered

carefully. Currently, emerging genetic engineering

techniques, combined with other therapies, like

adoptive T cell therapy (ACT) and CPI are applied in

OVs. These will make OV therapy one of the most

promising immunotherapies in the future (Zheng,

2019).

Xu, Y., Li, Y., Yin, Z., Jin, Z., Li, W. and Che, Z.

Use of Oncolytic Vaccinia Virus Armed with Certain Cytokines (IL-7, IL-12, IL-15, IL-21) in Combination with Checkpoint Inhibitors to Treat Non-Small Cell Lung Cancer.

DOI: 10.5220/0012032500003633

In Proceedings of the 4th International Conference on Biotechnology and Biomedicine (ICBB 2022), pages 457-466

ISBN: 978-989-758-637-8

 2023 by SCITEPRESS – Science and Technology Publications, Lda. Under CC license (CC BY-NC-ND 4.0)

457

1.2 General Functions of Cytokines and

CPIs in Regular and Antitumoral

Immune Responses

Cytokines are polypeptides or glycoproteins secreted

by diverse immune cells including T cells,

neutrophils, and macrophages and could regulate

immune responses (Cohen, 1996). Some cytokines

have been discovered to have potent anti-tumor

properties, which makes cytokine a monotherapy or

potentiator of other therapies in cancer treatment

(Berraondo, 2019). IL-7, IL-12, IL-15, and IL-21 are

chosen in this article. IL-7 is needed in B-and T-cells

development and could diminish cancer cell growth

(Alderson, 1991). IL-7 treatment was also reported to

enhance long-term CD8+ T-cell responses in mouse

model (Colombetti, 2009). IL-12 can activate effector

Th1 response, thus serving as a link between innate

and acquired immunity. This also further induces

activation of T-cell, NK-cells, and tumor clearance.

(Mirlekar, 2021; Zundler, 2015). IL-15 can promote

differentiation and expansion of T-cells, B-cells, and

NK cells, which leads to enhanced tumor response.

Moreover, IL-15 is important in the ontogeny of NK

and CD8+ cells (Isvoranu, 2021). IL-21 is involved in

co-stimulation of B-cell differentiation and

immunoglobulin production, stimulation of NK and

CD8+ cytotoxic function and co-mitogen of T-cells

(Sondergaard, 2009).

Checkpoint inhibitors (CPIs), on the other hand,

are important in enhancing T cell activation to combat

tumors (Zheng, 2019). T cell exhaustion,

characterized by loss of effector function and other

properties, arises during chronic exposure to antigens,

which limits tumor control (Wherry, 2011). Several

inhibitory pathways including PD-1 and PD-L1 play

important roles in this process. This led to the

development of CPIs to recover dysfunctional T cells,

including PD-L1 inhibitors, PD-1 inhibitors and

CTLA-4 inhibitors (Vaddepally, 2020). Nevertheless,

CPIs are ineffective for 'cold' tumors with low

infiltration of T cells. OV’s infecting and lysing the

tumor cells could improve intra-tumoral infiltration

and solve the limitations of CPI; thus, co-treatment of

OVs and CPI is a natural trend.

1.3 The Non-Small Cell Lung Cancer

Non-small cell lung cancer (NSCLC) is a

heterogeneous disease accounting for about 84% of

all lung cancer diagnoses in the United States

(Molina, 2008). Current treatment of NSCLC

includes surgeries, chemotherapy, radiation therapy,

and therapies targeting cell cycle control and

apoptosis (Molina, 2008). The immune checkpoint

inhibitors have been used recently to treat

unresectable stage III NSCLC, using anti PD-1/PD-

L1 antibodies (Onoi, 2020). Studies have also

reported the improved treatment of NSCLC with

several cytokines, such as IL-7 and IL-12. The

cytokine induced killer cells and chemotherapy can

effectively increase the overall survival of patients

with advanced stages of NSCLC. IL-7 can aid in the

sensitivity of NSCLC towards chemotherapy drug

cisplatin. IL-12 is also shown to directly target human

lung adenocarcinoma cells as well as adjacent normal

bronchial epithelial cells (NBEC) (Airoldi, 2009).

However, the primary and acquired resistance to PD-

1/PDL1 blockade mechanisms in NSCLC have been

reported, which might arise from components in the

immunosuppressive tumor microenvironments that

leads to inefficient activation and infiltration of T

cells (Pathak, 2020).

2 NEW TREATMENT

A new therapy called JX-594alpha is designed, which

consists of JX-594 (an oncolytic vaccinia virus) that

expresses IL-15, IL-12, IL-7, IL-21 and PD-L1

inhibitor (iPDL1). Oncolytic vaccinia virus (VV), JX-

594 is chosen as the delivery platform for several

reasons. VV, compared to other types of oncolytic

viruses, has a large genome size that allows it to

accommodate multiple foreign genes (Breitbach,

2013). This makes it possible to carry a combination

of the genes encoding IL-7, IL-12, IL-15, and IL-21.

VV also exhibits features such as rapid replication, a

wide tropism, and easy recombination for making

viral mutants (Hawkins, 2002). Besides, JX-594 with

granulocyte-macrophage colony-stimulating-factor

(GM-CSF) gene and deletion in thymidine kinase

(TK) gene could enhance immune responses and

selectively replicate in cancer cells with mutated RAS

or p53 genes (Merrick, 2009).

The cytokines (IL-7, IL-12, IL-15, IL-21) could

boost T-, B-, and NK cell performance. while CPIs

allow tumor recognition by T cells. Therefore, we

believe that these two subjects working in tandem

could substantially enhance antitumor effect. We

hypothesize that the JX-594alpha is able to initiate

antitumor immune responses that would eventually

lead to tumor regression in NSCLC mouse model.

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3 EXPERIMENT

3.1 Generation and Characterization

3.1.1 Generation of 4 Engineered OV

The generation of the JX-594alpha is achieved by

inserting genes encoding different ILs（including IL-

7, IL-12, IL-15, and IL-21) and iPDL1 into vaccinia

virus. During the experiment, five groups will be

generated, including VV-iPDL1/IL, VV-IL, VV-

iPDL1, VV-empty, and the control group. The VV

shuttle vector pSel-DsRed2N1 will be used to deliver

iPDL1 into the VV (Wang, 2020). The VV shuttle

vector pCMS1-IRES will be used to deliver the ILs

into the VV (Ge, 2020).

3.1.2 iPDL1 Expression and Secretion in

Infected H460 Cells

24 h after infection, supernatants of H460 cells are

collected. iPDL1 concentration will be detected by

mice iPDL1 ELISA kit in control, VV-iPDL1/IL,

VV-IL, VV-iPDL1, and VV-empty groups.

3.1.3 Characterization of iPDL1

Supernatants of the tumor cells infected with VV-

iPDL1/IL are harvested and then purify the

supernatant to get iPDL1. Then the binding of

purified iPDL1 to PD-L1+/+ or PD-L1 -/- cells is

determined. The infected cells are incubated with

purified iPDL1 or IgG before they are stained with

antibodies against PD-L1 or IgG Fc.

3.1.4 Expression, Secretion and

Characterization of IL

To test the expression and secretion of ILs in the

H460 cells, harvesting the culture supernatants is

planned to measure IL-7, IL-12, IL-15, IL-21 using

ELISA. The presence of these ILs will be observed in

the control, VV-iPDL1/IL, VV-IL, VV-iPDL1, and

VV-empty groups, respectively.

3.1.5 Cytotoxicity

To test cytotoxicity of the immune checkpoint, the

H460 cell line with KRAS mutation is used. Tumor

cells will be plated in well-plates. Two groups are

planned to be used to perform the experiment, with

each group containing 5 subgroups with varying gene

insertions. The PD-L1 +/+ tumor cells will contain

control, VV-iPDL1/IL, VV-IL, VV-iPDL1, and VV-

empty cells. The PD-L1 -/- tumor cells will be

generated using the CRISP-Cas9 technique and will

contain the same subgroups as that in PD-L1 +/+. The

cell viability will be determined 48 hours after

infection with cell-counting kit or nonradioactive cell

proliferation assay (Ge, 2020).

3.2 Antitumor Activity in Mice NSCLC

Model

3.2.1 Model Construction

The mouse mutant LSL-KrasG12D is proposed to be

used as our mouse model. This mouse model was

generated by Xu and others where they crossed mice

carrying CC10-CreER allele to Lox-stop-Lox (LSL)

K-RasG12D mice. They expressed oncogenic codon

12 mutant K-Ras in CC10- and Sftpc-expressing cells

in the adult mice lung using knock-in CreER driver

mouse lines. The researchers found that CC10+ type

II cells are one of the origins of adenocarcinomas in

response to K-Ras activation. The phenotype of the

mice shows adenomas and adenocarcinomas with

SPC-Cre, which works for our experiment to test the

oncolytic viruses in NSCLC (Xu, 2012).

3.2.2 OV Injection

Continuing from the last step, the antitumor activity

of the oncolytic virus is evaluated in the established

model. The mice are divided into five groups

randomly: control group, VV-iPDL1/IL, VV-IL, VV-

iPDL1 and VV-empty group. When their tumors

reach a volume of 100mm

(day 0), they receive intra-

tumoral injections of 50μL indicated VV three times

on 0,3,7 days post-transplantation and the control

group receive an equal amount of PBS at the same

time and site.

3.2.3 Characterization of iPDL1 and IL

After the previous step, the expression of iPDL1 and

IL levels in tumor-bearing mice are detected. Serum

samples are collected from mice without treatment,

injected VV-iPDL1/IL, VV-IL, VV-iPDL1 or VV-

empty 3 days after the infusion of indicated VV. The

serum levels are determined using mouse PD-1

ELISA kit and mouse IL-7, IL-12, IL-15 and IL-21

ELISA kit. To explore how long the iPDL1 could

maintain in tumor-bearing mice, both the serum

sample and tumor tissue are collected from the mice

1, 2, 4, 7, 10, 15, 20 …days after VV-iPDL1/IL

injection. iPDL1 level is determined using mouse PD-

L1 ELISA kit until iPDL1 level is too low to be

detected. The kinetics curve of iPDL1 level is made

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accordingly. There are three independent samples in

a group and each experiment is repeated twice.

3.2.4 Tumor Growth and Survival

The tumor growth is monitored after the injection of

VV to observe the progression of NSCLC. At 3, 7, 10,

13, 16, 20, 25, 30 days, each mouse in five groups

receives microCT scan, which is a relatively accurate

and easy method for tumor volume measurement

(Jensen, 2008). MicroCT images are analyzed by

certain softwares to get tumor volumes. Kaplan-

Meier survival curve is made within 100 days. There

are five independent individuals in a group.

3.2.5 Tumor Infiltration and Immune Cell

Ativation

Five days after VV injection, tumor tissues are

collected from mice without treatment, injected VV-

iPDL1/IL, VV-IL, VV-iPDL1 or VV-empty and

digested with collagenase type I and DNase. Then the

tissues are filtered to prepare single-cell suspensions,

which are then subjected to antibodies staining and

analyzed by FACS. Antibodies against CD45, CD8,

CD4, CD11c, CD11b and Gr-1 are used. Then the

plots are drawn based on the percentage of infiltrating

CD45+ immune cells, dendritic cell (DC; CD11c+),

Myeloid-derived suppressor cells (MDSCs;

CD11b+Gr-1+), CD4+ T cells, CD8+ T cells in tumor

tissues. Besides, we assume that the virus could also

activate the infiltrating effector T cells. The IFN-γ

and TNF-α expression of CD8+ T cells can be

measured by intracellular staining. There are five

independent samples in a group and each experiment

is repeated twice.

3.3 Anticipated Results

Below are the idealistic results of the experiments

based on conjecture.

3.3.1 Characterization of VV in H460 Cells

In our assumptions, high levels of iPDL1 can be

detected in VV-iPDL1/IL and VV-iPDL1 infected

H460 cells, while IL-7, IL-12, IL-15 and IL-21 levels

of VV-iPDL1/IL and VV-IL infected cells are

significantly higher than other groups. The pictures

are not shown here. Besides, it is hypothesized that

VV-iPDL1/IL-infected cells release a higher level of

iPDL1 than VV-iPDL1-infected ones and a higher

level of the interleukins than VV-IL-infected ones.

This could be explained by the synergy between the

interleukins and immune suppressive pathways.

Moreover, iPDL1 protein purified from the

supernatant of VV-iPDL1/ IL infected H460 cells

should bind to PD-L1+/+ tumor cells, but not to PD-

L1 -/- tumor cells in vitro. Expected results are shown

in figure 2. Flow cytometry is used to characterize

PD-L1 expression on different H460 cells first

(Figure 1). These could attest to the the successful

expression of iPDL1 that can specifically bind to PD-

L1+/+ tumor cells.

Figure 1: Expression of PD-L1 on PD-L1+/+ H460 cells and PD-L1 -/- H460 cells (Wang, 2020) Flow cytometry is used to

show the PD-L1 expression on both wild type H460 cells and PD-L1-knocked out H460 cells.

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Figure 2: iPDL1 secreted by infected H460 cells could bind to PD-L1 +/+ cells (Wang, 2020) PD-L1 +/+ and PD-L1 -/- H460

cells are incubated with purified iPDL1 or IgG (used as a negative control) before being stained with anti-iPDL1 or anti-IgG

antibodies. Flow cytometry shows the percentage of iPDL1 that binds to PD-L1 +/+ H460 cells.

3.3.2 Enhanced Cytotoxicity in H460 Cells

As is shown in figure 3, for the PD-L1 +/+ H460 cells,

the viability of the cells infected with VV-iPDL1/IL

should be lower than that of those infected with VV-

iPDL1, VV-IL and VV-empty (the cell viability of

control group is 1). As is shown in figure 4, for the

PD-L1 -/- H460 cells, although we hypothesize that

the overall cytotoxicity is higher due to the lack of

immune checkpoint pathway, there should be so

significant difference between VV-iPDL1/IL and

VV-IL, VV-iPDL1 and VV-empty. In conclusion, the

secretion of the iPDL1 from JX-594 alpha is assumed

to lead to cell killing, in a PD-L1 dependent manner.

Figure 3: Cell viability of wild type H460 cells infected with indicated VVs H460 cells are infected with VV-iPDL1/IL, VV-

IL, VV-iPDL1 and VV-empty respectively. Control group is incubated with the same amount of PBS. The cell viability is

determined 48 hours after infection using nonradioactive cell proliferation assay.

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Figure 4: Cell viability of PD-L1-knocked out H460 cells infected with indicated VVs CRISPR/Cas9 is used to generate PD-

L1 knocked out H460 cell lines. The following procedures are parallel to those in wild type H460 cells.

3.3.3 Characterization of VV in LSL-

KrasG12D Mouse Model

High levels of iPDL1 should be detected in mice

infected with VV-iPDL1/IL and VV-iPDL1, while

IL-7, IL-12, IL-15 and IL-21 levels in mice infected

with VV-iPDL1/IL and VV-IL group should be

significantly higher than other groups. In a word, the

anticipated results are similar to that of in vitro studies

and the figures are omitted here. Additionally, iPDL1

levels in both the tumor and serum of mice injected

with VV-iPDL1/IL are assumed to reach their peak

several days after injection and could last for about 20

days (Figure 5), which is of great clinical importance.

Figure 5: Kinetics of iPDL1 levels in both tumors and sera of the VV-iPDL1/IL-treated mice (Wang, 2020). iPDL1

concentrations are determined using PD-L1 ELISA kit 1, 2, 4, 7, 10, 15, 20 days after intra-tumoral injection.

3.3.4 Enhanced Antitumor Activities in

LSL-KrasG12D Mouse Model

As is shown in figure 6, we assume that tumors in

mice treated with VV grow slightly before shrinking

and the tumors all disappear finally. But the tumor

volume of mice treated with PBS should increase

substantially. Among the mice treated with VV, the

tumors disappear earliest in VV-iPDL1/IL group

within 30 days, the second is VV-iPDL1 group and

following are the VV-IL and VV-empty groups. As is

the survival curve in figure 7, we hypothesize that

mice in VV-iPDL1 group have the highest survival

rate 100 days after injection. These all demonstrate

that intra-tumoral injection of VV-iPDL1/IL could

significantly inhibit tumor growth in KrasG12D

mouse model although other VV are also potent

tumor inhibitors.

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Figure 6: Tumor volume of mice injected with indicated VVs within 30 days LSL-KrasG12D mice are intratumorally injected

with indicated VVs when their tumors reach a certain size. At 3, 7, 10, 13, 16, 20, 25, 30 days, each mouse receives microCT

scan to determine tumor volume.

Figure 7: Kaplan-Meier survival curve of tumor-bearing mice injected with indicated VVs within 100 days (Caroline, 2020)

LSL-KrasG12D mice are intratumorally injected with indicated VVs and monitored in 100 days. Each group has five

independent samples.

3.3.5 Enhanced Tumor Infiltration and

Immune Cell Activation

We hypothesize that FACS analysis (Figure 8) could

indicate a higher level of overall lymphocytes marked

by CD45+ and a higher percentage of CD4+, CD8+

T cells and dendritic cells in VV-iPDL1/IL group

compared to VV-IL and VV-empty group. For

MDSC cell, VV-IL and VV-iPDL1/IL should both

enhance its composition compared to PBS group.

However, we assume the MDSC cells in VV-

iPDL1/IL group doesn't increase as much as the other

two groups, indicating the ability of VV-iPDL1/IL to

inhibit immune suppressive cells. Besides, VV-

iPDL1/IL should activate effector T cells by

enhancing the expression of IFN-γ, CD107a and

TNF-α in CD8+ T cells (Figure 9). In a word, it's

speculated that VV-iPDL1/IL could enhance the

infiltration of lymphocytes, inhibit the suppressive

cells and activate CD8+ effector T cells.

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Figure 8: FACS analysis of infiltrating CD45+ immune cells, CD4+ T cells, CD8+ T cells, dendritic cells and MDSCs in

tumors (Wang, 2020) 5 days after VV injection, tumors are harvested and digested to prepare single-cell suspensions, which

are stained by antibodies against CD45, CD8, CD4, CD11c, CD11b and Gr-1.

Figure 9: Expression of IFN-γ, TNF-α, and CD 107a in infiltrating CD8+ T cells (Wang, 2020) Intracellular staining is used

to measure IFN-γ and TNF-α expression of CD8+ T cells.

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4 DISCUSSION

The JX-594 alpha has large genome size which

enables it to express multiple cytokines and

checkpoint inhibitors (CPI) and thus induce T-cell

proliferation and antibody production. With the help

of several cytokines with different functions, our

modified virus can increase immune response and

target tumor cells more efficiently. In our

expectations, the overall benefits of our virus are to

induce a regression of tumor size and increase

survival rates.

Despite the benefits of combination stated above,

our experiment has some drawbacks. First, five

subgroups are designed here: VV-iPDL1/IL, VV-IL,

VV-iPDL1, VV-empty, and the control group. We

would explore how the addition of iPDL1, cytokines

and the combination of both iPDL1 and cytokines

could affect the properties of VV. However, we

regard the four cytokines as a whole rather than

explore their functions separately. This might bring

about a question of whether or not the final increase

in immune responses arises from the synergy of

cytokines. It’s possible that one or two cytokines

don’t act as immune activating agent in this condition.

Thus, more detailed studies on the function of each

cytokine in VVs are needed. Besides, oncolytic virus

therapy could be combined with other therapies in

further studies. For instance, a study has investigated

the combination effect of ReoT3D and some

chemotherapeutic agents in NSCLC cells and

demonstrated that ReoT3D and taxane could achieve

synergy through apoptosis (Sei, 2009). To make the

virus more aggressive to cancer cells, we may also

start to look for other proteins besides cytokines and

CPIs that also increase immune responses. Finally,

genetic modifications of VVs could enhance their

antitumor effect. However, some properties of viruses

might be impaired at the same time like their stability

and safety, which can cause some unexpected side

effects.

5 CONCLUSION

Based on previous studies of OV therapy, a new

remedy, JX-594 alpha is conceived and its

effectiveness in NSCLC is tested in both H460 cell

lines and KrasG12D mouse model. We hypothesize

that both VV-iPDL1 /IL and VV-iPDL1 infected

H460 cells could produce iPDL1 and the levels of IL-

7, IL-12, IL-15 and IL-21 in VV-iPDL1 /IL infected

cells were higher than those in other groups. Besides,

iPDL1 produced in vitro should be able to bind PD-

L1 +/+ tumor cells, which is tested by flow cytometry.

VV-iPDL1 /IL is also assumed to cause cell killing in

a PD-L1-dependent way. As to the in vivo

experiment, it’s also hypothesized that constructed

VVs could produce iPDL1 or the cytokines

successfully in mouse mode l. The long-term level of

iPDL1 in both the serum and tumor is also measured.

Tumor growth and overall survival of the mice are

monitored within 100 days. It is expected that the

tumor disappeared earliest in VV-iPDL1 /IL group,

and the survival rate of VV-iPDL1 /IL group is also

the highest. Other VVs should also be potent tumor

suppressors, but weaker than VV-iPDL1 /IL. FACS

should be used to characterize tumor infiltration and

immune cell activation. We assume that the VV-

iPDL1 /IL group has a higher overall CD45+

lymphocyte level and a higher percentage of DC,

CD4+ and CD8+ T cells. In addition, VV-iPDL1 /IL

should activate effector T cells by enhancing IFN-γ

and TNF-α expression. In conclusion, JX-594 alpha

is assumed to enhance lymphocyte infiltration,

activate CD8+ effector T cells and finally achieve the

goal of tumor elimination in NSCLC mouse model.

Admittedly, the experiment design has some

drawbacks discussed above. But we believe that

emerging studies on OV therapy will definitely bring

it to clinical application.

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