Developing CAR-T Therapy for Treating B Cell Malignancies

Clara Xi Wang

1,†

, Haoyang Guo

2,*,†

, Hanqin Yang

and Beibo Kang

College of Arts and Sciences, New York University, New York, NY, 10003, U.S.A.

Eberly College of Science, Pennsylvania State University, University Park, PA, 16802, U.S.A.

Jinan Foreign Language School, Jinan, Shandong, 250108, China

BASIS International School Guangzhou, Guangzhou, Guangdong, 510663, China

Keywords:

CAR-T, PD-1 Inhibitory Receptors, Cytokine Release Syndrome, CD40 Ligand, T Cell Type Ratio.

Abstract:

B cell lymphoma is a type hematopoietic malignancy with an average incidence rate of 4.3%. While B cell

lymphoma is not as deadly as other solid tumors, aggressive lymphomas such as the diffuse large B cell

lymphoma (DLBCL) can be fatal due to its fast-spreading characteristic and high metastatic ability. To more

effectively target B-cell lymphomas, scientists have recently created the chimeric antigen receptor (CAR) that

can actively detect the CD19 antigens secreted by cancer cells and directly activate T cells without binding to

the major histocompatibility complex (MHC). However, over time, many cancer cells have also developed

several mechanisms to escape the detection of T cells and to inhibit their function, which can significantly

hamper the overall efficacy of traditional CAR-T therapy. Furthermore, traditional CAR-T therapies may also

cause severe side effects, such as the cytokine release syndrome (CRS) caused by an overproduction of

proinflammatory cytokines. In this study, we examined six current research articles that address these immune

escape mechanisms as well as the side effects caused by traditional CAR-T therapies. We propose an

experimental CAR-T therapy that combines the major findings from this primary research, which, if proven

feasible, can substantially improve the overall efficacy of CAR cancer immunotherapy while significantly

reducing damage caused by side effects.

1 INTRODUCTION

B cell lymphoma is a hematopoietic malignancy

characterized by the proliferation of abnormal B

lymphocytes (Swiner, 2020). Most cases of B cell

lymphoma belong to the category of non-Hodgkin

lymphomas (NHL) such as the fast growth DLBCL

and the indolent chronic lymphocytic leukemia

(CLL). CLL generally grows much slower than

aggressive types, but they are also less curable with

standard treatments and, if left untreated, can

potentially grow into a more aggressive form of

cancer. Additionally, even though 40-50% of all

patients with DLBCL can achieve complete

remission after therapy, 30-40% of patients relapse

within a short time and 10% develop refractory

DLBCL, a type of cancer that does not respond to any

type of treatment. Patients with relapsed/refractory

DLBCL are less responsive towards conventional

Corresponding author.

†

They are both first authors

cancer therapies, and even if receiving second

treatments with higher doses of chemotherapy and

stem cell transplants, r/r DLBCL patients have a 1-

year survival rate of only 28% (Raut, 2014). These

characteristics make B cell lymphoma more

dangerous compared to other cancers. As a result,

there exists a dire need for new cancer therapies that

specifically target B cell lymphoma.

Current cancer treatment options include surgery,

chemotherapy, and radiation therapy, as well as the

latest techniques such as the minimally invasive

interventional radiology and immunotherapy.

However, because chemotherapies may cause serious

side effects such as non-specific cytotoxicity and

generalized immune suppression, scientists have

recently been investigating different

immunotherapies that utilize the immune system

itself to offer long-term remission through

immunological memory. Among different cancer

immunotherapies, CAR-T therapy that involves the

Wang, C., Guo, H., Yang, H. and Kang, B.

Developing CAR-T Therapy for Treating B Cell Malignancies.

DOI: 10.5220/0012015100003633

In Proceedings of the 4th International Conference on Biotechnology and Biomedicine (ICBB 2022), pages 149-155

ISBN: 978-989-758-637-8

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

149

manipulation of T lymphocytes in the adaptive

immune system has shown significantly greater

efficacy against B cell lymphoma in multiple

research studies and clinical trials.

1.1 CAR-T Therapy

The activation process of T lymphocytes involves the

interaction between the T cell receptor (TCR) and

specific antigens presented by the MHC as well as

several co-stimulation factors such as CD28, CD137,

and OX40. However, to evade T-cell detection tumor

cells have developed several immune inhibitory

functions, such as upregulating inhibitory molecules

as well as reducing MHC expression (Yilmaz, 2020).

CAR-T therapy addresses these limitations by

creating T cells that function independently of MHC

molecules (Graham, 2018). CARs are split into three

domains: ectodomains, a transmembrane domain,

and an intracellular domain of CD3ζ for signal

transduction (Figure 1). The ectodomain is the most

vital and different from traditional TCRs; it consists

of a signal peptide and the antigen recognition

domain of the single-chain Fragment variant (ScFv)

derived from the F

region of antibodies fused by a

linker.

Figure 1: Structure of a Chimeric Antigen Receptor.

The Ectodomain consists of the linker, spacer,

ScFV head, and signal joint. The single-chain

variable fragment (ScFv) is responsible for the

specific antigen recognition from CAR cells.

Composed of the Fab regions of light and heavy

chains of an immunoglobulin, the ScFV is connected

to the CAR via a short linker peptide. The spacer, also

known as the hinge region, connects the antigen

recognition region to the surface of the membrane. It

enhances ScFV flexibility and promotes the binding

of recognition regions to target cells (Guedan, 2019).

Advantages of CAR-T arises from the fact that

they are MHC-independent, which allows it to

recognize any type of surface antigen, including

carbohydrates and lipids. Additionally, as memory T

cells remain in circulation long-term, the benefits of

CAR-T therapy can last for several years. CAR-T

therapies have little time for treatment, as it consists

of a single injection after which the patient can be

released after two weeks. Because the treatment itself

is not as aggressive as chemotherapy and radiation,

patients tend to have a much more rapid recovery.

Additionally, CAR-T can be a final resort for patients

who do not qualify for stem cell transplantation or

suffer from multiple relapses.

1.2 Side Effects of CAR-T Therapy

To date, CAR-T cells have shown remarkable

antitumor activity in patients because of the long-

lasting remissions in hematologic malignancies that

are not responding to standard therapies. For

example, the CAR-T therapy Axicabtagene

Ciloleucel can reach a tumor objective response rate

of 82% and a complete response rate of 54% (Levine,

2016). However, CAR-T therapies also have serious

limitations, such as the CRS. CRS is a severe side

effect of CAR-T therapies, in which the signalling

mechanism involved can provoke secretion of

cytokine and activation of macrophages (Neelapu,

2017). As a result, while investigating mechanisms to

decrease this side effect in CAR-T therapies,

researchers have also started to incorporate natural

killer (NK) cells and nanobodies in CAR-Therapies.

2 PRIMARY RESEARCH

STUDIES

To resolve different immune escape mechanisms

developed by tumor cells, we first examined two

papers, which introduced the idea of engineering

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CAR-T cells to make CAR receptors actively secrete

anti-PD-1 antibody and CD40 ligand (CD40L).

Consequently, we evaluate the efficacy of the IL-6

binding protein in reducing the CRS discussed in the

third study. To maximize the overall effectiveness of

CAR-T therapy, we further examined the effector

function of different subsets of T cells discussed in

the fourth paper.

2.1 Engineered CAR-T Cells with

Self-Secreting Anti-PD1 Antibodies

Have Shown Optimistic Results in

Suppressing PD-1 Inhibitory

Receptors in the Tumor

Microenvironment (TME) of

CD-19 Expressing Tumors

CAR-T treatments’ long-term efficacy has been

impeded by the upregulation of several cell surface

inhibitory molecules in the TME, such as cytotoxic T

lymphocyte-associated protein (CTLA-4) and

programmed death-1 (PD-1). These inhibitory

receptors can negatively regulate the proper

activation of T cells by competing with the co-

stimulator CD28 for the B7 ligand. Without proper

activation, the overall immune response generated by

the adaptive immune system will be largely

hampered (Li, 2017). Among different inhibitory

molecules, the upregulation of PD-1 in CAR-T cells

can cause not only hypofunction of CAR-T cells but

also dysfunction of tumor infiltrate cells (TLC)

following specific antigen stimulation. As a result, in

this study, scientists aimed to combine the CAR-T

therapy with the PD-1 blockade mechanism to

overcome the inhibitory effect of PD-1. They

conducted the experiment using lung cancer line

NCI-H292 and designed a special CAR-T cell,

CAR19.alphaPD1, targeting CD-19 antigen by

inserting a gene fragment that can express the ScFv

from anti-PD1 antibody using retroviral vector. To

test whether CAR19.alphaPD1 can reduce the

immune inhibitory effect caused by PD-1,

researchers performed a competitive binding and

blocking assay on both CAR-T and

CAR19.alphaPD1 cells. In this experiment, T cells’

activity after being stimulated by anti-CD-3

antibodies was measured by intracellular IFN gamma

in the assay. According to the results, after the

recombinant human PD-L1 ligand were applied to

each well in the assay, the IFN gamma level

significantly, but this reduction of IFN gamma count

was quickly reversed after the addition of

CAR19.alphaPD1, indicating a successful blockade

of PD1 receptor by the self-secreted anti-PD-1

antibody. In addition, although PD-1 was upregulated

in both CAR-T and CAR19.alphaPD1 following

antigen stimulation, researchers in this study have

also discovered that expression of PD-1 was

significantly lower in CAR19.alphaPD1 T cells

compared to parental T cells, which can further

reduce the inhibitory effects caused by PD-1 in the

TME. Furthermore, in multiple other tests on

CAR19.alphaPD1’s ability to enhance antigen-

specific immune response, antitumor reactivity, T

cell proliferation, and T cell effector function,

CAR19.alphaPD1 in these tests all showed improved

efficacy compared to the control.

Based on the results, CAR19.alphaPD1 has

proven to be more effective in targeting H292-CD19

bearing tumors as well as reducing the immune

inhibitory signal in the TME compared to parental

CAR-T cells. Overall, CAR19.alphaPD1 can be

considered as an effective way to target CD19-

expressing tumors in future cancer immunotherapies.

2.2 Engineered CAR-T Cells with the

Ability to Secrete CD40L Can

Effectively Prevent Tumor Immune

Escape Caused by Antigen Loss

Tumor immune escape can occur via antigen loss so

that CAR-T cells lose their targets. To overcome

these negative impacts, CAR-T cells are engineered

to constitutively express CD40L to increase the

activation of the CD40/CD40L pathways in B cell

lymphoma. CD40 receptors are located on the surface

of abnormal B cells in B lymphoma. By transiently

activating CD40, CD40L can direct anti-proliferation

and apoptosis signals to cancer cells, thus effectively

reducing the outgrowth of B lymphoma (Odorizzi,

2012).

In addition, the CD40/CD40L pathway is highly

utilized for the activation of APCs, such as dendritic

cells (DC). The recruited APCs can further initiate

antitumor T cell responses by activating CD4

and

CD8

as well as secreting IL-12, which functions by

inhibiting the suppressive function of immune-

inhibitory macrophages, enhancing the antitumor

response of CAR-T cells, and recruit other non-CAR-

T cells at the same time (Elgueta, 2009).

Because the CD19

tumor can escape lysis by

traditional anti-CD19 CAR-T cells used in this study

(m1928z), the effectiveness of engineered CAR-T

cells with CD40L (m1928z-CD40L) on CD19

tumor

cells needs to be investigated. In this research study,

scientists modified the CD19

A20 lymphoma cell

line with the green fluorescent protein (GFP) and co-

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151

cultured CD19

GFP

A20 cells with both m1928z

CAR-T cells and m1928z-CD40. The results showed

that by day 21, the outgrowth of CD19

tumor cells

can be successfully eliminated by m1928z-CD40L

compared to m1928z, indicating that m1928z-

CD40L is more effective in detecting antigen-

negative tumor cells in the long term through the

increased activation of the CD40/CD40L pathway.

(Elgueta, 2009).

2.3 CAR–T Cells with Mbail6 Have

Complete Antitumor Activity and

Neutralize Macrophage-Derived

IL-6, Which Could Prevent CRS

The CRS has been one of the most severe side effects

of traditional CAR-T therapy since the development

of the first generation of CAR-T cells.

Pathophysiologically, CRS is mediated by

proinflammatory cytokine interleukin-6 (IL-6)

mainly secreted by activated macrophages (Kuhn,

2019). Patients with CRS experience acute systemic

inflammatory responses characterized by fever,

fatigue, and headaches, which can be life-threatening

in some cases. Currently, the IL-6 receptor inhibitor

tocilizumab has been approved by FDA to treat CRS,

yet its effect is not stable. In this study, researchers

use a non-signaling membrane-bound IL-6 receptor

(mbaIL6) that possesses anti-CRS activity while

maintaining CAR-T cells’ complete antitumor

capacity. Compared with the control groups, which

are Jurkat cells with only GFP expression, mbaIL6-

expressing Jurkat cells showed a significant

reduction of IL-6 concentration in cell culture. The

neutralization of IL-6 by mbaIL6 is further tested

using the U937 cell line, in which stimulation is

dependent on IL-6-mediated STAT3

phosphorylation. When cocultured with mbaIL6-

expressing Jurkat cells, STAT phosphorylation

reaction was significantly reduced compared to the

control group. These results imply a successful

neutralization of IL-6 by mbaIL6, which is critical in

reducing CRS in CAR-T therapies.

Consequently, researchers engineered traditional

CAR-T cells using a bicistronic MSCV vector

containing genes encoding mbaIL6 and anti–CD19-

41BB-CD3z CAR. The mabIL6 expression in

peripheral blood T lymphocytes was proved to have

no interference with the immunophenotype of T cells.

Further experiments have also shown that the

expression of mabIL6 on anti-CD-19 CAR-T cells

does not affect their normal proliferation rate,

suggesting that a combination of anti-CD-19 CAR

and mabIL6 could be a frontline treatment for B-

lymphoid malignancies and multiple myeloma

(Neelapu, 2017).

2.4 Using a Defined Subset of CD8+

TCM and CD4+ TN Cells in a 1:1

Ratio Has Synergistic Antitumor

Effects in CAR-T Therapy

Traditional CAR-T therapy involves the injection of

CD3

CAR-T cells with nonspecific ratios of CD8

and CD4

subsets, leading to varying frequencies of

CD8

and CD4

T cells in all patients. Due to this

discrepancy, it is difficult to set a consistent baseline

for gauging the effectiveness of CAR-T therapy as

well as maximizing the positive effects of CAR-T. In

this study, subsets of T cells were tested for their

respective antitumor efficacy to determine the best

combination of specific T cells to maximize CAR-T

efficacy.

In this study, CD8

and CD4

were first tested for

subset efficacy and then for the combined effect of

superior CD8

and CD4

subsets on murine B cell

lymphomas in a set ratio (Sommermeyer et al., 2016).

CD8

and CD4

subsets were tested respectively in

NOD SCID IL2RgNULL (NSG) mice. The NSG

mice engrafted with CD19

Raji tumors then tested

for cytokine release, cytolytic abilities, and survival

grafts, with mice grafted with EGFRt-T cells serving

as control. The results revealed a hierarchy of subset

effector functions. CD8

and T

cells showed

more cytolytic ability while CD4

cells had

superior cytokine release. Given that CD8

and

CD4

had superior antitumor responses, doses of

either CD8

, CD4

, or both of them combined

in a 1:1 ratio were administered to the mice. Mice that

received a 1:1 of CD8

and CD4

showed

better survival rates as well as more complete tumor

eradication, judging from the bioluminescence

imaging data and survival curve. This data leads to

the conclusion that certain defined T cell subsets do

enhance antitumor response, in this case, a 1:1

combination of CD8

and CD4

This study is significant because the antitumoral

efficacy of CAR-T cell subsets has never been

previously studied, and it is revealed that subsets of

CD8

and CD4+ CAR-TN have the strongest

antitumoral effects. Moreover, this study

demonstrates combining CD8+ TCM and CD4+ TN

cells in a 1:1 ratio has a synergistic antitumor effect

than exclusively using one subset (Sallusto, 1999).

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3 DISCUSSION

Upon further examination of current research studies,

we hypothesized a new experimental CAR-T therapy

to maximize CAR-T efficacy while reducing side

effects. This new CAR-T therapy will incorporate all

the modification processes shown in previous

research studies, including the self-secreting anti-PD-

1 CAR, the ability to secrete CD40 ligands and the

IL-6 binding proteins all administered in a 1:1 CD8

to CD4

cells (figure 2), which, if proven to

be successful, can significantly increase the efficacy

of CAR immunotherapy while reducing the potential

side effects to the lowest level.

Figure 2: Cell model in the modified CAR-T cancer therapy.

CAR-T cells that target CD19 antigen (CAR19)

will be engineered to express anti-PD-1 antibody

(Anti-PD-1), CD40 ligand (CD40L), and membrane

binding IL-6 receptors (mbaIL6). These new proteins

are engineered on both CD8-expressing central

memory T cells (CD8

) and CD4-expressing

naive T cells (CD4

), The ratio of the two types of

T cells will be manipulated to reach 1: 1 in this new

therapy.

To successfully create this new CAR-T therapy

targeting B cell malignancy as well as to test the

proper expression of different receptors and their

effectiveness, we propose several experiments using

different methods and analyzed potential results that

could reflect whether this new treatment is feasible

for future cancer therapies.

3.1 Gene Insertion During CAR-T

Engineering

The insertion of genes of anti-PD-1, CD40L, and

mbaIL6 achieved by introducing retroviral vectors

into T cells. The retroviral vector can accommodate

genes of interest and incorporate its genes into the

target cell genes (Hambach, 2020). It has been largely

used during CAR-T cell engineering because of its

high transfer efficiency as well as its variety of gene

expression based on different types of viruses used

(Guedan,, 2019). In our proposed experiments on

CAR engineering, the retroviral vector of MP71 will

be used to insert the ScFv of the anti-PD-1 antibody

derived from human mAb 5C4 (Habib, 2019); MSCV

vector will be used as the basis to transduce

membrane bond anti-IL-6 derived from human anti-

IL-6 monoclonal antibody AME-19a (Neelapu,

2017); finally, the SFG-m1928z-CD40L will be

constructed using Gibson Assembly (Elgueta, 2009)..

3.2 Testing Successful Expression of

Engineered Receptors Using

Western Blot

The successful expression of multiple receptors in

our proposed CAR-Ts can be detected using western

blot, a technique used to identify specific proteins of

interest through the binding of specially designed

antibodies. After binding to these designed

antibodies, proteins of interest will be stained and

visualized through gel electrophoresis. Different

types of proteins in a protein mixture will be

separated based on their molecular size (Kurian,

2020). Based on previous studies, it has been found

that CD40L has a molecular weight between 32 to 39

kDa (Odorizzi, 2012), while anti-PD-1-producing

CAR has a molecular weight of approximately 27kDa

(Habib, 2019). Since these two proteins have a

significant weight difference, antibodies designed

separately for these two proteins can be added

together in western blot using the original CD19

CAR as the control group. If the gel electrophoresis

result shows two distinct stains, one located on 27

Developing CAR-T Therapy for Treating B Cell Malignancies

153

kDa and the other between the range of 32 to 39 kDa

on the newly designed CAR-T cells while no clear

stain for the control group, it can be indicated that

CD40L and anti-PD-1-producing CAR has been

successfully expressed. If no stains are shown, or

they indicate different molecular weights, then the

engineered protein may not be successfully

expressed.

3.3 Detection and Isolation of Target

T cells Using Flow Cytometry

Assay

In this experiment, we aim to isolate CD8

central

memory T cells and CD4

naïve T cells based on their

surface marker proteins using flow cytometry assay.

As naïve T cells predominantly express CD62L

(Sallusto, 1999), we designed antibodies specifically

targeting CD4 and CD62L. After the flow cytometry

assay, cells showing higher affinity to both CD4 and

CD62L will be collected and used as the naïve T cell

in the therapy. Because central memory cells are have

CCR7+, the remaining memory T cells expressing

CCR7 will be further isolated using anti-CCR7,

completing isolation of CD4+ TN and CD8+ TCM.

After extracting T

and T

cells, we propose to

further isolated T cells that simultaneously express all

three receptors engineered on the new CAR-T cell

using fluorescence-activated cell sorting (FACS).

The target T cells are identified and extracted based

on their binding intensity to antibodies designed for

the three different proteins. Based on previous

research, we have determined it is best to administer

the CD8

cells and CD4

cells in a 1: 1 ratio,

which has shown to have the greatest synergistic

antitumor effect (Sallusto, 1999). Therefore, after

isolating the target T cell subsets, we will manipulate

a 1: 1 CD8

CAR-T

to CD4

CAR-T

ratio before

conducting experiments in vivo.

3.4 Testing mbaIL-6’s Effective

Reduction of CRS

Infusion of CAR-T cells could lead to potentially life-

threatening CRS, which would sharply increase the

cost related to this treatment. Based on previous

experiments of mbaIL6, we decided to insert gene

encoding mbaIL6 to our newly designed ultimate

CAR-T cells to stay one step ahead of current studies

by combining mbaIL6 with other improvements on

CAR-T cells. Transduction of T lymphocytes could

be done with a construct that allows simultaneous

expression of mbaIL6, CAR, anti-PD1, and CD40L

to approach more effective antitumor capacity

without the presence of CRS. This construct could be

placed on an MSCV retroviral vector containing

genes of mbaIL6, anti-CD 19 CAR, anti-PD1, and

CD40L. For precise detection of anti-CD 19, CD19-

myc will be connected to the extracellular domain of

the CD19 molecule, and cells are stained with the

CD19-myc fusion protein and an anti-myc antibody.

Through variable control, degree of expression of

mbaIL6, CAR, anti-PD1, and CD40L, cell marker

profile (CD4, CD8, etc.), the proportion of naive,

effector, central memory, and effector memory, and

level of IL-6 Neutralization performed by mbaIL6

can be observed. To test whether IL-6 neutralization

by mbaIL6 can interfere with antitumor capacity

activated by CAR, we allow T cells to coculture with

CD19

OP-1 ALL cells and measure IFN-γ

production by flow cytometry assay after labeling

with anti-human IFN-γ–PE. The Level of cytotoxic

granules released by ultimate CAR-T cells can be

tested by staining with the anti-CD107a antibody.

The xenograft models could be used to determine the

in vivo antitumor capacity of ultimate CAR-T cells,

CD191 ALL cell line Nalm-6 IV (a B cell precursor

leukemia cell line) will be injected in NSG mice

along with ultimate CAR-T with and without

mbaIL6. Furthermore, levels of human IL-6

neutralization in vivo can be determined through

injecting ultimate CAR-T with and without mbaIL6

accompanied by IP injection of human IL-6 several

days later. If this combination works in xenograft

models, we would see considerable antitumor

capacity with and without expression of mbaIL6 on

ultimate CAR-T for a relatively long period.

Significantly, ultimate CAR-T expressing mbaIL6

can neutralize mbaIL6 to a lower level compared

with ultimate CAR-T not expressing mbaIL6 and

prevent CRS (Neelapu, 2017).

3.5 Testing T Cell Efficacy Through

Cytolytic Ability and Survival Rate

of Engrafted Mice

After injecting our proposed CAR-T therapy in the

NSG mice models, the efficacy of our modified

CAR-T therapy, we propose to use chromium release

assay as a measurement of cytolytic activity and

survival grafts to detect survival rates of mice bearing

CD-19 B lymphomas. Mice receiving only EGFRt-T

cells will serve as the control. If this combined

treatment were effective, we would expect to see a

significant increase in the survival rates and cytolytic

abilities of mice receiving the modified CAR-T

therapy compared to those receiving the control.

However, if the chromium release assay and survival

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rates show no significant difference or show a

significant decrease between the modified CAR-T

group and the control, we can deduce that our

modified CAR-T therapy fails to have a synergistic

effect on tumor masses.

4 CONCLUSION

In this study, we looked into six current research on

various CAR cancer therapies. Based on the methods

and results shown in these research experiments,

proposed a new CAR-T therapy model that combines

anti-PD1 and CD40L secretion, mbaIL6 receptor. To

further improve the efficacy of this new model, we

also suggested an optimal 1:1 CD8 T

to CD4 T

cell ratio. The proposed experiment will be tested in

vivo using the NSG mice model, which, if proven to

be successful, can significantly improve the overall

CAR-T cell efficacy in treating B cell lymphoma.

However, because we are unable to conduct actual

experiments, the feasibility and overall efficacy of

this newly designed CAR-T therapy will have to be

thoroughly investigated in future experiments.

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