Combination Therapies Increase the Efficacy of Melanoma

Treatment with Reduced Side Effects

Haoming Zhang

, Jiayi Zong

, Jingyi Guo

, Lawrence Ma

, Yijia Chen

and Zhaojun Qiu

6,*

College of Animal Sciences and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou,

Guangdong 510025, China

Jinling High School, Nanjing, Jiangsu 210029, China

Beijing National Day School, Beijing 100039, China

Ridge High School, Basking Ridge, New Jersey 07920, U.S.A.

School of Life Science, Fudan University, Shanghai 200433, China

Shanghai Pinghe School, Shanghai 201206, China

lawrencema0512@gmail.com, yijiachen18@fudan.edu.cn,

qiuzhaojun@shphschool.com

Keywords: Immune Checkpoint Inhibitor, Kinase Inhibitor, Oncolytic Virus, Combinational Therapy.

Abstract:

T Immunotherapy is found to have a promising effect on cancer treatment. It initiates activation of the immune

response to fight against cancer. Previous studies have demonstrated that immune checkpoint inhibitors (ICIs),

kinase inhibitors, and oncolytic viruses (OV) are possible cancer immunotherapies. Immune checkpoint

inhibitors targeting programmed cell death-1 (PD-1) and programmed death-ligand 1 (PD-L1) have achieved

great success in cancer immunotherapy. We hypothesized that utilizing anti-PD-1 antibodies, mitogen-

activated protein kinase (MAPK) inhibitors, and oncolytic virus (T-VEC) could boost the efficacy of

traditional PD-1 therapy towards melanoma, while the side effects can be alleviated with the supplement of

anti-cytokine antibodies and anti-inflammatory drugs. A series of experiments were designed to be conducted

in melanoma murine models. Expected outcomes of this combination therapy include enhanced tumor

regression, extended survival, and mitigated side effects. The success of this study could bring up a new

strategy for melanoma therapies.

1 INTRODUCTION

1.1 Melanoma

Melanoma is a major type of skin cancer that arises

from genetic mutations in melanocytes, the pigment-

producing cell, which can be found throughout the

skin, eye, inner ear, and leptomeninges. Although it

only takes up a small percentage of all malignant skin

cancer, it is the most aggressive and deadliest type.

As shown in Figure 1, Once it becomes metastatic

(move to other organs from where it originated), the

prognosis is very poor (Domingues, 2018), needing

better treatments to be studied and applied to.

1.2 Theraputical Mechanism in the

Proposal

As shown in Figure 2, T cells, recognizing peptide

antigen with the aid of cell surface major

histocompatibility complex (MHC) molecules, have

two broad classes with very different functions,

named by their expression of CD4 or CD8 co-

receptor: CD4+ T cells detect the antigen in MHC

class II molecules and act as the headmaster in the

adaptive immune system by producing cytokine,

chemokine, and pro-inflammatory responses. While

CD8+ T cells detect antigen with MHC class I

molecules and carry out direct toxicity to kill infected

or cancerous cells (Darvin, 2018).

104

Zhang, H., Zong, J., Guo, J., Ma, L., Chen, Y. and Qiu, Z.

Combination Therapies Increase the Efﬁcacy of Melanoma Treatment with Reduced Side Effects.

DOI: 10.5220/0012014600003633

In Proceedings of the 4th International Conference on Biotechnology and Biomedicine (ICBB 2022), pages 104-115

ISBN: 978-989-758-637-8

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

Figure 1: The four stages of melanoma (Biorender. 2021).

Figure 2: CD8+ T cell activation by Dendritic cell and effects on cancer cell (Biorender. 2021).

Generally, the interaction between multiple

checkpoints and regular stimulatory signals regulates

T cells in an effective but not autoreactive fashion

(Sharpe, 2018). But tumor cells can abnormally

utilize this signal system in two ways – reduced

stimulatory signals or overexpressed checkpoint

signals. Immune checkpoint inhibitors (ICIs) can

target the overexpressed checkpoint signals that allow

tumor cells to evade immunosurveillance, and

function by releasing the natural breaks on immune

activation and enhancing the T-cell immune ability to

eliminate tumor cells (Darvin, 2018).

The proposal mainly focuses on the druggable

inhibitors of the T cell PD-1 (Programmed cell death-

1) pathway and its complementary ligand (PD-L1).

As shown in Figure 3, PD-1 is a transmembrane

inhibitory receptor, and signals through its pathway

are mainly responsible for controlling initial T cell

activation as well as the effecting function of the cell.

On the other hand, PD-L1 is located on cancer cells

binds to the PD-1 inhibitor, and it tricks the T cell

from functioning (Sharpe, 2018). Antibodies have

been engineered to specifically target either PD-1 or

PD-L1, the former takes off the brakes from T cells,

and the latter prevents cancer cells from "hiding". It

is also noteworthy that their feasibility has been

further proved by structural analysis (Lee, 2016).

Combination Therapies Increase the Efﬁcacy of Melanoma Treatment with Reduced Side Effects

105

Figure 3: PD-1-PD-L1 interaction and its inhibition strategy.

Left: The schematic model of PD-1-PD-L1

interaction in a tumor cell. The binding suppresses the

activation of T cell.Right: The antibody (ICI) binds to

PD-1, therefore, inhibits the interaction which leads

to T cell activation and induces apoptotic death of

tumor cell.

Tumors can evolve to evade both innate and

adaptive arms of the immune system, thereby

rendering ICI therapy ineffective (Pfirschke, 2016;

Mueller, 2015). A subset of patients receiving

immune-checkpoint inhibitor therapy develop

unconventional response patterns (termed 'pseudo-

progression') that can be misinterpreted as disease

progression (Nishino, 2017). The MAPK pathway

provides significant therapeutic targets due to its

aberrant activation in cancer and strong interference

with complex molecular pathways, leading to wider

use of MAPK inhibitors in melanoma, lung cancer,

colorectal cancer, and other types (Smith, 2014;

Germann, (2017). Permanent activation of RAS

protein caused by mutations accounts for a very high

proportion of all human cancers through activating

downstream signaling pathways, including the

MAPK family (the downstream of RAS GTPase,

represented by RAF and its variant), then the MEK

family (MAP kinase-ERK kinase), and

ERK1/2(Extracellular signal-regulated kinases),

specifically shown in Figure 4. Activated Therapies

that target RAS-activating pathways or RAS effector

pathways could be combined with these direct RAS

inhibitors, immune checkpoint inhibitors, or T cell-

targeting approaches to treat RAS-mutant tumors

(Moore, 2020; Braicu, 2019).

Figure 4: The more detailed and specific schematic demonstration of the RAS/MAPK pathway (Biorender 2021).

Yet some MAPK pathway effectors (e.g., p38α)

play a dual role, with suppression in one cancer but

inflammation in another cancer, making preclinical

studies more considerable (Grossi, 2014). In

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melanoma, MAPK inhibitors are mainly resisted by

macrophage-derived TNF-α (Smith, 2014), lowering

down the efficacy and implying the possibility of

adverse effects. To further strengthen the

development of MAPK inhibition, complementary

combination therapies can be added to raise the

efficacy.

Figure 5: The mechanism of oncolytic virus against tumors (Biorender 2021).

Viruses can be used in attacking tumor cells (Bai,

2019). The left panel illustrates the oncolytic viral

response to normal cell vs malignant cell. The middle

panel illustrates a general mechanism of the oncolytic

virus with T cell activation at the tumor site. The right

panel illustrates transgene expression in oncolytic

virus.

Oncolytic viruses (OVs) are treated as natural or

engineered viruses that replicate specifically in

cancer cells and kill them. The mechanism of their kill

process is shown in Figure 5. They are harmless to

normal organisms (Fukuhara, 2016) and able to be

delivered both systemically and locoregionally. Thus,

they can act at the primary or metastatic tumor sites

(Twumasi-Boateng, 2018).

According to Figure 6, T-VEC is a type of OV for

melanoma that consists of a double-mutated oncolytic

herpes simplex virus type 1 (HSV-1) armed with

granulocyte-macrophage colony-stimulating factor

(GM-CSF) (Biorender 2021). It was approved

officially as the ﬁrst oncolytic virus drug by the US

Food and Drug Administration (FDA) on 2015,

October 27th (Pol, 2016).

Though T-VEC is designed to be injected locally

into the tumor, systemic effects are often induced,

leaving a body free of cancer, but with flu-like

symptoms. Besides, due to the potential for viruses to

attack healthy cells, the risk of infection still exists

(Marelli, 2018).

Figure 6: The mechanism of T-VEC oncolytic virus to aid T cell against tumor cell (Biorender 2021).

Combination Therapies Increase the Efﬁcacy of Melanoma Treatment with Reduced Side Effects

107

2 FORMULATION OF

HYPOTHESIS

2.1 Combination of Different

Orthogonal Therapeutic

Approaches

For an optimistic therapeutic effect to be reached, a

combinational therapy consisting of checkpoint

inhibitors (PD-1, PD-L1), as well as oncolytic viruses

(T-VEC) may be adopted. Studies have previously

demonstrated the improved efficacy combined

therapies are likely to yield, details of which will be

introduced later as this article progresses. Recently,

therapies of OV combined with ICI have been

uncovered. The oncolytic virus can secrete PD-L1-

secreting-OVs. It systemically cleaves and inhibits

PD-L1 on tumor cells and immune cells, initiating

tumor neoantigen-specific T cell responses. Tumor-

specific oncolytic immunotherapies for cancer

treatment have been made available to patients

(Wang, 2020).

Furthermore, a triple combination of OV, ICI, and

kinase inhibitors is proposed and compared with a

dual combination of OV and ICI or kinase inhibitors

and ICI in mice models. The triple combination is

consisting of a phosphatidylinositol 3-kinase (PI3K)

inhibitor, a PD-L1 inhibitor, and VSVΔ51, which is

an engineered OV strain from vesicular stomatitis

virus (VSV), commonly is used as a therapy for

PTEN-deficient glioblastoma. The result shows that

dual combination enhances immune response

slightly, while triple combination therapy increases

the treatment efficacy significantly. Tumor regression

is induced accompanied by complete tumor

eradication in most mice that are treated with triple

combination therapy. Long-term antitumor immune

memory is established in these mice (Xing, 2021).

2.2 Management of Side Effects

ICIs’ ability to stimulate the immune system has

further contributed to them being appreciated as a

desirable treatment for cancer (Simonaggio, 2019).

However, they also lead to toxicities and several kinds

of inflammation in different organs. Frequently

affected organs include the skin, and organs in the

digestive, and endocrine systems (Durrechou, 2020).

New research has found that the mechanism of action

of ICIs reveals a new toxicity profile called immune-

related adverse events (irAEs) (Richmond, 2008).

Recurrences (both same types and different types)

of irAE after recovery is another difficult problem

(Martins, 2019). Currently, the commonly used PD-

1/PD-L1 monoclonal antibody has the mechanism of

removing immunosuppression and activating T cell

function. T cells are also found in normal tissue.

While it is killing tumor tissue the side effects may

occur at the same time.

The co-stimulation of T-lymphocyte leads to

inflammatory cytokines. When the body meets

inflammation, the immune system will secrete many

inflammatory cytokines, which regulate lots of

aspects of cell growth and differentiation and play a

key role in the coordination of immune defenses

against invading. They are potentially immunogenic

which could generate the anti-cytokine

autoantibodies (aCA) (Meager, 2014). A significant

portion of the anti-inflammatory cells express PD-L1

by inducing target cells, and then T lymphocytes kill

the target cells.

With low irAE grade, ICI was discontinued

follow-up, and steroids were used for grade 2 or

higher adverse events. The core principles of

management included continuing treatment with ICI,

early detection and adequate control of irAEs may

contribute to improved patient prognosis (Matsuoka,

2020). High-risk patients receiving ICIs should be

regularly monitored for associated complications by

a professional multidisciplinary team, preferably

using a personalized monitoring strategy (Martins,

2019).

2.3 Mini Review

The purpose of the proposal is to solve the problem

that how to increase the therapeutic effect and range

of ICI-associated therapy while decreasing its side

effects. Based on previous findings among three

kinds of therapies and other related research, we have

eventually chosen to focus on melanoma and formed

a hypothesis: utilizing the combination between ICI

(PD-1 and PD-L1 antibodies), MAPK inhibitor, and

oncolytic virus (Talimogene laherparepvec) can

effectively increase the therapeutic effect of

melanoma treatment, while the side effects can be

reduced by anti-cytokine antibodies and anti-

inflammatory drugs.

Through the design of experiments in mice

models and the estimated results, we expect to shed

light on the triple combination immunotherapy in

melanoma treatment and provide our views on ideal

therapies with high efficacy and low adverse effects.

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EXPERIMENTAL

APPROACHES

In order to test the hypothesis, there will be two major

experiments in this study – one is to test the effect of

combinational therapy, one is to test the reduction in

side effects with anti-cytokine antibodies and anti-

inflammatory drugs.

3.1 Experimental Object

3.1.1 A Brief Introduction to Xenograft

Tumor on Humanized Mice

In order to examine a patient’s tumor’s response to a

certain therapy, research must examine with human

tumor instead of mouse generated tumor (Richmond,

2008). To do so human tumor xenografts can be

implanted subcutaneously into immunosuppressed

mice, so the mice acquire human tumor cells to

establish tumor microenvironment and propagation

inside the mice. This is a critical model for various

studies on cancer interaction and behavior with the

cardiovascular and immune systems and response to

the various drugs (Martins, 2019).

3.1.2 Processes of Mouse Model Generation

Hetero transplantation of human cancer cells or tumor

biopsies into immunodeficient rodents as patient

xenograft models) (PDX)constituted the major

preclinical screen for the development of novel

cancer. the models have identified clinically

efficacious agents and make effort in pharmaceutical

industry therapeutics (Morton, 2016). In order to

make mouse models, several processes are needed.

SCID is mice with defective combinations of T, B,

and NK cells. First, the il2R γ KO mice were

backcrossed after the deletion of the IL-2 receptor γ

chain gene, which is a common domain of cytokine

receptors. Then, NOD/SCID−γ−/−c mice are

irradiated (sublethal, 1 Gy, whole-body irradiation)

(Gonzalez, 2013), followed by CD34+ stem cells

from the same cord blood donor each with a unique

UCB donor was performed. In all cases, recipient

mice were evaluated for human hematolymphoid

engraftment at 12 to 16 weeks post-injection. For

new-boring mice, human hematolymphoid cells

(HSC) engraftment of newborn mice by intracardiac

(IC) injection can be better (Brehm, 2010).

Meanwhile, subcutaneous implantation of autologous

human thymic tissue (1–2 mm3 fragments) could also

work (Shankar, 2020). Followed by the establishment

of in vitro human cell lines to be propagated

subcutaneously, reconstituting solid tumors (Russell,

2018).

3.1.3 Advantages and Disadvantages of the

Processed Mouse Models

As shown in Figure 7, the feature complexity of

genetic and epigenetic abnormalities that only present

inhuman compared to mice. The results can be

obtained relatively rapidly (a few weeks), and

multiple therapies can be tested from a single tumor

biopsy. Although the humanized mice can partially

reconstruct the human immune system, the process to

generate humanized mice is tedious and costly (Gong,

2018).

Figure 7: The advantages and disadvantages between three common types of mouse models in cancer research (Russell, 2018).

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109

3.1.4 Control Factors

All the humanized mice are expected to have same

age and gender. They are treated with same

environmental condition (25 Celsius, same water and

food). All the Xenograft tumor is taking from the

same human patient. The xenograft human bone

marrow is derived from HLA compatible healthy

participant. Every kind of drug are produced in same

company in similar date.

3.2 Experiment for Combinational

Therapy

In this experiment, we will test out the efficacy of ICI

and ICI combination therapy by utilizing different

combinations of treatment on controlled mice models.

3.2.1 Positive and Negative Control

The positive control in our experiment is humanized

mice without tumor xenograft to test out the natural

rate of death and normal symptoms as a comparison

to the experimental groups. The negative control is

tumor xenograft humanized mice without any

treatment in order to test out the effect of advanced

melanoma on the mice without any outer interference.

3.2.2 General Protocol

Step 1 – humanized mice cultivation (see Figure 8)

Figure 8: The schematic diagram about the first step in the experiment protocol.

Prepare 72 humanized mice (4-6 all male) and

stratified into six sub-groups evenly (n=12) by

randomization. Inject healthy individual’s (HLA

matched with tumor xenografted patient)

hematopoietic stem cell into the mice and wait at least

12 weeks to let the immune system become mature.

After 12 weeks, examine blood sample from 2-3 mice

in each group by flow cytometry to estimate amount

of immunological cell in the mice. If it reaches the

expected standard, then start Step 2.

Step 2 - Prepare and culture xenograft advanced

melanoma tumor from the human patient (see Figure

9).

First, extract tumor tissue sample from an

advanced melanoma patient, who is HLA-matched

with the hematopoietic stem cell donor. Then

cultivate it under ideal environment until reach

enough amount for transplantation. Mince the

proliferated tumor tissue into equal pieces that are

sufficient to cause melanoma in the mice in a short

amount of time while not lethal immediately. Last,

Inject the equivalent minced tissue into the

humanized mice by subcutaneous injection.

Figure 9: The second step of the experiment is where cultures and transfer of human melanoma tumors are performed.

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Step 3 - Inject the corresponding treatment to

different humanized Mice groups (see Figure 10)

Wait 4 weeks after tumor injection in order to let

the tumor spread in the mice. Then, inject

corresponding treatment intravenously to each group

of mice.

Figure 10: The lab protocol demonstrates the experiment phase where injection of treatments and observations are taking

place.

Step 4 - Observation and record

Observe the mice for at least 2 months. The

purpose and method to acquire data will be presented

in the following section.

3.2.3 Technology and Index for Result

Measurement

Both macroscopic observation and data (number of

mice dead and clinical symptoms) and microscopic

and data (T cell density and activation status) are

necessary to support studies about combination

therapy. In macroscopic observation, the survival

curve and clinical statistics can be obtained (see

Figure 11). Kaplan Meier (KM-Curve) is a great

estimation of how the model survives during long-

time observation.

Figure 11: The figure shows how the Kaplan Meier graph is obtained by counting the number of survived and dead mice

models in each group every day.

3.2.4

Anticipated Results

Survival rates of mice, average tumor size and T cell

density are main index being expected to be

measured, to evaluate the efficacy of each treatment

through macro and micro approaches.

Negative control (no treatment):

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111

Negative control group is expected to have

Poorest Kaplan-Meier (K-M) curve that has the

steepest slope (which means many mice dead in a

short amount of time). It also expected to have largest

average tumor size and most tumors with least

number of lymphocytes and myeloid cells. Also, it is

expected to have least amount of fluorescence (least

amount of activated T cell against tumor cell)

ICI (PD-1) only:

With only ICI treatment only, the mice expected

to have a better and flatter K-M curve compared to

negative control. The tumor size and density should

have smaller average tumor size and reduced number

of tumor (despite pseudo-progression). It should have

more active (higher fluorescence density)

lymphocytes compared to negative control.

ICI with MAPK (RAS inhibitor):

As expected, double treatment should result in

better K-M curve, smaller average tumor size and

density, and more and active lymphocytes compare to

ICI treatment only and negative control.

ICI with the oncolytic virus (T-VEC):

The result obtained with ICI and T-VEC double

treatment expected to similar with ICI and MAPK

double treatment without significance differences

(p>0.05).

Triple combination (ICI + T-VEC + kinase

inhibitors):

The triple combination will be expected to have

best K-M curve, least tumor size and density, and

most numerous and active lymphocytes (with tumor).

Positive control (no tumor): normal lifespan

without injection

The K-M curve should follow natural rate of

death, and with no tumor observed. Also, there should

be normal number of lymphocytes and normal

activation status since no infection or cancer takes

place.

ICI treatment can initially increase tumor size,

then suppress the tumor size at later date, as expected

with pseudo-progression (see Figure 12). The

negative control group is a mice model that grafted

with a tumor but does not receive any kind of

treatment. The positive control group is a mice model

that is normal (no tumor graft).

Figure 12: The average tumor size of each experimental group.

3.3 The Experiment of Management of

Side Effects

3.3.1 Positive and Negative Control

The positive control in this experiment is healthy

mice without xenograft tumors. The negative control

is tumor xenograft humanized mice with the triple

combination but does not have any medicine to

manage side effects.

3.3.2 Anticipated Results

In the ideal state, the same number of the humanized

mice in the four groups have both symptoms cause by

ICIs. Here we use DCR (disease control rate) to

describe the anti-irAE efficacy through each

treatment. Overall survival rate is expected to be

measured to characterize disease control and is

demonstrated in Figure 13.

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Figure 13: The overall survival (OS) curve of experimental groups. OSR stands for Overall Survival Rate.

The negative control is a mice model with

melanoma xenograft tumor that only treated with

combinational therapy (without any anti-side effect

treatment). The positive control is normal mice with

a natural rate of death. The triple combination with

anti-IL6 antibodies performs worse than

corticosteroids because anti-IL6 antibodies reduce the

therapeutic effect of the triple combination.

4 DISCUSSION

4.1 Advantages and Limitations for

Melanoma

Combination with a variety of drugs can be induced

by a different mechanism and thus increase antitumor

immune response, leading to improvements in the

overall curative effect. Despite the humanized mice

more closely mimicking diverse populations

reconstruct the human immune system, discrepancies

still exist. Triple therapy might be incompatible with

anti-cytokine inhibitors such as by reducing or even

abolishing the effects.

4.2 Possibilities of Triple Combination

Therapy in Other Cancers

As hundreds of clinical trials have been conducted on

malignancies that have a relatively dramatic response

to PD-1 and PD-L1 blockade, nine cancer types have

been approved for the PD-1/PD-L1 treatment (Gong,

2018). Among them, MAPK inhibitors and oncolytic

viruses were seen significant activity in non-small

cell lung cancer (NSCLC), Renal cell carcinoma

(RCC), and Hepatocellular carcinoma (HCC) in

previous studies (Baines, 2011) Therefore, we can

presume the possibilities of such a triple therapeutic

modality applying for these kinds of cancers. Xing F

et al have made efforts on the combination of anti-

PD-1 treatment, kinase inhibitor (PI3K), and OV and

testified that its efficacy was synergistically and

safely restored in PTEN-deficient GBM models

(Matsuoka, 2020). Under the support that ICIs, kinase

inhibitors (MAPK), and OV each have positive

responses in these cancer types, meanwhile, dual

combinations among these therapies have enhanced

the effects, we can similarly expect higher

effectiveness of therapeutic responses by utilizing our

triple combination therapy. Facing the challenge of

irAEs caused by ICI therapies, we launched our

solutions that using inflammatory cytokines

inhibitors or corticosteroids collaboratively to reduce

the irAE grade. For cancers of NSCLC, RCC, and

HCC, the same levels of irAEs compared with

melanoma were detected (Shankar, 2020); Baines,

2011; Ornstein, 2017). After triple combination

therapy is used to raise efficacy, anti-irAE strategies

(anti-cytokine and corticosteroids) may also be able

to be applied.

ACKNOWLEDGMENT

All authors contributed equally to this work and

should be considered co-first authors.

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