CAR-T cell therapy: precise targeting, promising future

Chimeric antigen receptor (CAR)-T cell therapy is a revolutionary new pillar of cancer immune cell therapy, which has shown significant clinical treatment effects in patients with B-cell leukemia or lymphoma, but CAR-T therapy is helpless in the face of solid tumors, and CAR-T therapy also has serious life-threatening toxicity and limited anti-cancer efficacy
.
In this article, we will introduce the latest research progress of CAR-T cell therapy, the limitations of CAR-T cell therapy, and potential improvement strategies
.

In recent years, the field of targeted tumor therapy has shown a vigorous development trend
.
Tumor immunotherapy has attracted much attention because of its advantages of strong specificity and small side effects, and has gradually entered the public's field of vision
.
The emergence of tumor immunotherapy has changed the development pattern
of cancer treatment.
Immunotherapies
such as cytokine therapy, vaccines, immune checkpoint inhibitors, and adoptive cell therapy have been reported.
Among them, CAR-T cell therapy, as the "sharp weapon" of the anti-cancer battlefield, has emerged suddenly, providing a new treatment option
for tumor targeted therapy.

Chimeric antigen receptor T cell immunotherapy belongs to a type of adoptive cell immunotherapy (ACT), and its family also includes TIL, TCR-T therapy, CAR-NK and other methods
.
CAR-T therapy refers to a new type of anti-cancer therapy in which T cells are collected from the patient's blood by apheresis, genetically engineered to express CAR and expanded in vitro culture, and then adoptively infused the CAR-T cells back into the patient (Figure 1).

Chimeric antigen receptor T cells (CAR-T cells) refer to a class of immune cells that conjugate the antigen binding part of an antibody that can recognize a tumor antigen with the CD3-ζ chain or the intracellular part of FcεRIγ in vitro as a chimeric protein, and transfect the patient's T cells
through gene transduction.
CAR is mainly composed of three functional domains: extracellular domain, transmembrane domain and intracellular domain (Figure 1).

At present, a total of four generations of CAR-T cells have been developed, of which the third and fourth generations of CAR-T technology are still in the research and development stage; The most widely used in clinical practice are the second-generation CAR-T cells, and the second-generation CAR-T technology is used in the CAR-T therapy
that has been marketed.

CAR-T cells are actually equivalent to installing the positioning and navigation device CAR on T cells, transforming ordinary T cells into "super soldiers", and using its positioning and navigation device CAR to specifically recognize tumor cells in the body, release immunoactive substances by activating the immune response, produce immune effects, thereby efficiently killing tumor cells, and then achieving the effect of treating malignant tumors (Figure 2).

Chimeric antigen receptor CAR-T cell therapy is revolutionary because it produces an effective and long-lasting clinical response
.
CAR binds to target antigens expressed on the cell surface and is independent of MHC receptors, activating T cells and generating a potent anti-tumor response
.
However, CAR-T cell therapy still has limitations that must be addressed, including life-threatening CAR-T cell associated toxicity, limited efficacy against solid tumors, inhibition and drug resistance of B-cell malignancies, antigen escape, limited persistence, limited transport capacity and tumor invasion capacity, and immunosuppressive microenvironment
.

1 Antigen escape

The biggest challenge of CAR-T cell therapy is tumor resistance to monoantigen-targeted CAR (Figure 3).

Although the initial monoclonal antigen-targeted CAR-T cells showed a high response rate, a large proportion of patients treated with these CAR-T cells showed antigen escape phenomena
such as partial or complete loss of target antigen expression.

One study found that although 70-90% of patients with relapsed or refractory ALL (acute lymphoblastic leukemia) showed a durable response to CD19-targeting CAR-T cell therapy, resistance developed in clinical use, and 30-70% of patients experienced disease recurrence
after treatment.

Similarly, downregulation or absence
of BCMA expression was observed in multiple myeloma patients receiving CAR-T cell therapy targeting BCM.
In addition, similar antigen escape resistance has
been observed in solid tumors.
To reduce the recurrence rate of CAR-T cell therapy for hematological malignancies and solid tumors, many strategies now rely on developing CARS
that target multiple antigens.
Current clinical trials using dual-targeted CAR-T cells (CD19/CD22 or CD19/BCMA) have also shown positive results
.

2 Target extraneoplastic effects

Studies have shown that solid tumor antigens are also often expressed at different levels on normal tissue, and CAR that targets solid tumor antigens may also target normal cells, causing unnecessary toxicity (Figure 4).

Therefore, antigen selection is crucial in CAR design: not only to ensure therapeutic efficacy, but also to avoid the occurrence
of "targeted extratumor" toxicity.
In addition, potential strategies to address targeted extratumoral effects are targets for tumor-restrictive post-translational modifications, such as truncated O-glycans overexpressed in solid tumors
.

3 Limited CAR-T cell trafficking and tumor invasion capacity

Compared with hematologic malignancies, CAR-T cell therapy for solid tumors is limited by the ability of CAR-T cells to transport and infiltrate solid tumors because physical tumor barriers such as the immunosuppressive tumor microenvironment and tumor stromitium limit the penetration of CAR and the movement of T cells (Figure 5).

Studies have shown that topical administration can improve these defects
.
Because local administration avoids the need for CAR-T cells to transport to the disease site and reduces the targeted detumor effect, CAR-T cells only target tumor cells, which can minimize interaction
with normal tissues.
In addition, preclinical studies have confirmed the significant efficacy
of intraventricular injection of CAR-T cells targeting HER2 and IL13Ra2.

4 Tumor immunosuppressive microenvironment

In the tumor microenvironment, many cell types that drive immunosuppression can infiltrate solid tumors, including myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and regulatory T cells (Tregs).

These cells can promote tumor production of cytokines, chemokines, and growth factors, which affect the infiltration of CAR-T cells, which in turn affects the efficacy of CAR-T cells (Figure 6).

In addition, studies have shown that immune checkpoint pathways such as PD-1 or CTLA-4 reduce anti-tumor immunity
.
Therefore, the combination of CAR-T cell therapy and immune checkpoint inhibitors is considered to be the next frontier
of immunotherapy.
Although combination therapy with immune checkpoint inhibitors and CAR-T cell therapy may be a new immunotherapy option, it is important to note that this combination therapy may not be sufficient to induce T cell infiltration and effector function
.

5 CAR-T cell-associated toxicity

CAR-T cell therapy is a revolutionary cancer treatment tool, but its high toxicity and some lethality rates prevent CAR-T cell therapy from becoming a first-line therapy (Figure 7).

Studies have shown that the key factors determining the incidence and severity of CRS, HLH/MAS and/or ICANS are the design of CAR, specific targets, and tumor type
.

At present, the toxicity of CAR-T cell therapy has been demonstrated in patients who received the first FDA-approved CAR-T cell therapy, and even in the most significant clinical trials, serious life-threatening toxic events
have occurred in patients.
In patients with acute lymphoblastic leukemia / lymphoma (ALL/LBL) receiving CAR-T cell therapy, almost all patients have at least some mild toxicity, and about 23-46% of patients show severe supraphysiological cytokine production and a large number of in vivo T cell expansion
.
However, there is currently no effective strategy to prevent the above toxicity, therefore, it is necessary to optimize the CAR structure and adopt other strategies to reduce the toxicity
caused by CAR.

To achieve an effective therapeutic response, the CAR-T cell antigen-binding domain must bind to its target epitope and reach the lowest threshold level for
inducing CAR-T cell activation and cytokine secretion.
CAR-T cells must remain within their treatment window to be clinically effective, as exceeding the treatment window can produce cytokines and immune system activation, which can lead to toxic side effects
.
Therefore, it is crucial
to optimize CAR-T cells to improve therapeutic effect and limit toxicity.

1 Transform and optimize the CAR structure

One way to reduce toxicity is by changing the affinity
of the antigen-binding domain of CAR-T cells.
Reducing the affinity of the antigen-binding domain leads to an increased need for higher antigen densities on tumor cells to achieve high levels of activation
.
Therefore, targeting healthy tissues with low expression of antigens can be avoided by reducing antigen affinity
.
In addition, the secretion
of CAR-T cell cytokines can also be regulated by modifying the hinge region and transmembrane region of CAR.

Studies have shown that optimizing the hinge and transmembrane regions may be useful ways to reduce toxicity, and a phase I clinical trial found that modifying CAR in the hinge and transmembrane regions resulted in complete remission
in 54.
5% of patients with B-cell lymphoma.
Similarly, the CAR-T co-stimulatory domain can also be modified according to tumor type, tumor burden, antigen density, and target antigen-antigen binding domain to reduce toxicity
.

2 Reduces CAR immunogenicity

The recognition of CAR by the body's own host immune system may lead to cytokine-mediated related toxicity
.
Therefore, the use of human or humanized antibody fragments instead of murine CAR to reduce CAR immunogenicity may be an effective strategy
to reduce virulence.
In addition, the immunogenicity of CAR can also be reduced by modifying hinges and/or transmembrane domains, and the persistence of modified CAR-T cells is also improved
.

3 Modified CAR-transduced T cells

The most common side effects of CAR-T therapy are cytokine release syndrome (CRS) and neurotoxicity
.
CRS refers to the release of a large number of cytokines in the process of immune cells and tumor cells, and these cytokines will trigger further chain reactions, such as excessive inflammatory reaction, capillary leakage, coagulation cascade, etc.
, which will lead to organ damage and brain swelling, and ultimately life-threatening
.
Studies have shown that modifying CAR-transduced T cells helps reduce the occurrence of CRS and reduce neurotoxicity
.

4 CAR "off switch" policy

Another potential way to improve CAR-T cell toxicity is through the implementation of "off switch" or suicide gene strategies
.
But the biggest limitation of suicide strategies or other similar methods is that, despite their attractiveness in ensuring safety, their use can suddenly stop treatment
of rapidly developing diseases.
This method can temporarily inhibit CAR-T cell function and can rescue CAR-T cell therapy
after the toxicity subsides.
In the future, it is necessary to develop more effective strategies that can temporarily inhibit CAR-T cell function and carry out CAR-T cell therapy rescue after the toxicity subsides, which is crucial
for CAR-T cell therapy to move towards the first-line treatment of hematology.

In recent years, with the continuous heating up of the field of CAR-T cell therapy, many domestic and foreign pharmaceutical companies have deployed CAR-T therapy, including pharmaceutical giants such as Novartis, BMS, and Gilead, and domestic pharmaceutical companies such as JW Therapeutics, Legend Biologics, Fosun Pharma, and Innovent have also participated in the layout
.
So far, there are 7 CAR-T therapies approved for marketing in the world, among which Kymriah is the world's first CAR-T therapy product developed by Novartis, which was approved by the US FDA in August 2017 for the treatment of children and adolescents with
acute lymphoblastic leukemia (ALL).
In addition, there are currently more than 300 CAR-T therapies in clinical trials
.

CAR-T cell therapy brings hope to patients with hematological tumors, especially in the treatment of patients with advanced malignancies
.
However, due to the problems of tumor immunosuppressive microenvironment, antigen heterogeneity, off-target toxicity, etc.
, the development of
CAR-T therapy is hindered.
Despite the challenges, new strategies and potential solutions are evolving and may provide the way forward for more effective and safer anti-cancer therapies
.
In addition, the high cost of CAR-T treatment is also an insurmountable mountain
.
It is believed that with the continuous research of CAR-T therapy, more safe and effective CAR-T therapies will be approved for marketing to benefit
patients.

References

       1、T cell immunotherapy for human cancer.
Science.
2018, 359, 1361–1365.

       2、Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma.
N.
Engl.
J.
Med.
2017, 377, 2531–2544.

       3、Engineering CAR-T cells: design concepts.
Trends Immunol.
2015, 36, 494–502.

       4、Engineering strategies to overcome the current roadblocks in CAR T cell therapy.
Nat.
Rev.
Clin.
oncol .
2020,17, 147–167.

       5、CAR T cell therapy in B cell acute lymphoblastic leukemia.
Sci.
Transl.
Med.
2014, 6, 224-225.

       6、A safe and potent anti-CD19 CAR T cell therapy.
Nat.
Med.
2019, 25, 947–953.