Pioneering CAR-T Cell Therapy: Advancing Personalized Cancer Treatment

1. Advances in CAR T-Cell Therapy Research

The cancer cell therapy field is expanding rapidly, particularly in CAR-T cell research over the past three years (2020-2022) ^[1]. However, most therapies are still in early development stages, with a broad range of cell sources being explored. The CAR-T cell therapy industry shows strong momentum but is still largely pre-commercial, indicating both high potential and ongoing scientific challenges.

1.1 What Is CAR-T Cell Therapy?

Chimeric antigen receptor T (CAR-T) cell therapy is a type of immunotherapy that harnesses the power of our immune system to fight cancer.

CAR-T cell therapy involves collecting blood from the patient to harvest T-cells, which are modified to express a CAR that recognizes and binds to specific tumor antigens. These engineered T cells are multiplied in the lab and infused back into the patient, where they identify the cancer cells with target antigens and kill them.

1.2 Development of CAR-T Cell Therapy

CAR-T cell therapy has evolved over the decades. Scientists first proposed the idea of using T cells with synthetic receptors in the late 1980s. But it wasn’t until the last 15 years that technology allowed researchers to make it happen safely.

In 1987, the concept of double-chain chimeric T-cell receptor (cTCR) was proposed by Yoshikazu Kurosawa ^[3], followed by the first description of the first generation of CARs with scFv in 1993 ^[4]. Key co-stimulatory domains CD28 and 4-1BB were incorporated into T cells through the CAR construct in 2002 and 2004, respectively ^[5,6]. The first CAR-T trials in patients began in 2006 ^[7,8]. The third generation, containing two or more costimulatory domains, emerged in 2010, and the fourth generation was developed in 2012.

By 2011, leukemia patients achieved complete remission with second-generation CAR-T. In 2012, Emily Whitehead became the first pediatric patient treated with CAR-T. The FDA approved CD19 CAR-T cell therapy (Kymriah and Yescarta) for leukemia in 2017. In 2021, Abecma was approved for multiple myeloma ^[9].

1.3 The Clinical Success of CAR-T Cell Therapy

In 2017, the FDA approved the first CAR T-cell therapy for children and young adults with acute lymphoblastic leukemia (ALL). Soon after, approvals expanded to aggressive lymphomas and multiple myeloma.

Table 1. FDA-approved CAR-T cell therapies for B cell malignancies and multiple myeloma ^[10]

(Note: R/R, relapsed or refractory; ALL, Acute Lymphoblastic Leukemia; DLBCL, Diffuse Large B-cell Lymphoma; FL, follicular lymphoma; MCL, Mantle Cell Lymphoma; LBCL, Diffuse Large B-cell Lymphoma; MM, Multiple Myeloma; B-ALL, B-cell Acute Lymphoblastic Leukemia)

However, CAR-T is more than a cancer treatment. It’s also a symbol of innovative biotechnology, and it has become a springboard for new therapies in autoimmune disease and infectious disease, too.

2. Key Parameters in CAR Design & Challenges and Limitations

2.1 Key Parameters in CAR Design

A CAR is a recombinant receptor that can reprogram T-cell function to target a specific antigen in cancer cells. It is composed of four parts, each of which contributes to the proper activation, functionality, and persistence of CAR-T cells.

① the extracellular antigen recognition domain of the single-chain fragment variant (scFv) derived from an antibody

• Affinity and avidity of ligand-binding domain
• scFv misparing and aggregation
• Antigen epitope location and accessibility

② the hinge region (or spacer domain)

• Link the scFv component and the transmembrane domain
• Contain protein sequences coming from molecules such as CD8α, TCRβ, and IgG
• Lend flexibility to the scFv and help improve efficacy

③ the transmembrane domain

• Anchor the CAR to the T-cell membrane
• A potential to offer different functionality and modularity to CARs

④ the intracellular domain

• Contain the T-cell activation domain of CD3ζ and the signaling domain of costimulatory receptors, which varies depending on the generation of CAR-T
• The different numbers and types of costimulatory molecules give rise to five generations of CARs

Conventionally, T cell activation usually involves two signals: the first signal is initiated when the T-cell receptor (TCR) interacts with peptide-loaded major histocompatibility complex (pMHC), and the second signal comes from costimulatory receptors like CD28. The less-than-optimal effectiveness of conventional T cells in killing cancer cells may be partly caused by the absence of CD80/86 expression on tumor cells.

Differently, synthetic CARs can bind to antigens directly tumor-associated antigens (TAAs) on the surface of cancer cells through the scFv domain rather than relying on immunogenic processing and peptide presentation by MHC receptors, thus enhancing the applicability of CAR-T to different patients ^[11,12].

2.2 Five Generations of CAR-T Cells

CARs have evolved. Currently, there are five generations of CAR-T cells.

Table 2. Five generations of CARs ^[13]

2.3 Killing Mechanisms of CAR T-Cells

How do CAR T-cells kill cancer? CAR-T cells mediate tumor killing through three axes:

① Perforin/Granzyme axis: When CAR-T cells recognize and bind to target tumor cells on the surface of cancer cells, they release perforin, which forms pores in the tumor cell membrane in a calcium-dependent manner. These pores allow granzymes to enter the tumor cell cytosol. Once inside, granzymes cleave key intracellular proteins, triggering apoptosis in the tumor cell. This pathway is particularly effective in targeting antigen-positive tumor cells.

② Fas and FasL axis (Death Receptor Pathway): CAR-T cells express FasL, which binds to Fas receptors on the surface of tumor cells. The Fas/FasL interaction activates the extrinsic apoptosis pathway, leading to the formation of the death-inducing signaling complex (DISC) and subsequent activation of caspases, which execute the apoptotic program in the tumor cell. This mechanism is particularly useful in cases where tumor cells may be resistant to other forms of cell death.

③ Cytokine secretion: Upon activation, CAR-T cells release cytokines such as IFN-γ, TNF-α, and IL-2, which directly induce apoptosis in tumor cells, enhance the cytotoxic activity of other immune cells, and promote an inflammatory microenvironment that is hostile to tumor growth. Additionally, cytokine production can recruit and activate other immune cells, amplifying the anti-tumor response.

2.4 Potential Hot Targets of CAR-T Cells

The success of CAR-T therapy largely depends on identifying specific antigens expressed on cancer cells, which serve as "hot targets" for engineered T-cells to attack.

Hematological tumor:

• Antigens associated with multiple myeloma, including CD19, CD38, CD138, BCMA, SLAM7, NKG2D, and GPRC5D
• Others including CD22, CD20, CD123

Solid tumor:

• Highly expressed in tumor tissue and low or absent in normal tissue, including MUC1, B7H3, HER2, GPC3, GD2, EGFR, MSLN, CLDN18.2 and CLDN6.

3. Challenges and Limitations of CAR-T cell Therapy

Although CAR-T cell therapy represents a transformative approach to cancer treatment, several challenges and limitations hinder its broader application and efficacy.

3.1 Challenges and Limitations

① Antigen Escape

Antigen escape occurs when tumor cells downregulate or lose the target antigen, rendering the therapy ineffective. This phenomenon is a significant barrier, as it allows cancer cells to evade immune detection and continue proliferating. Strategies to address this include targeting multiple antigens or developing CAR-T cells with broader specificity.

② On-Target Off-Tumor Effects

Many targets are not only on tumor cells. Even tiny amounts on normal tissue can be a problem. On-target off-tumor effects arise when CAR-T cells attack healthy tissues expressing the same antigen as the tumor. This can lead to severe organ damage and other adverse effects. For example, targeting CD19 in B-cell malignancies can also affect normal B cells, causing B-cell aplasia.

Antigen selection is closely correlated with sufficient anti-tumor efficacy and minimized toxicity concerns.

③ CAR-T Cell Trafficking and Tumor Infiltration

Effective CAR-T cell therapy requires the engineered cells to migrate to and infiltrate the tumor site. However, solid tumors often present physical and biological barriers, such as dense extracellular matrix and abnormal vasculature, which impede CAR-T cell trafficking. Improving CAR-T cell homing and penetration into tumors remains a critical area of research.

④ Immunosuppressive Microenvironment

The tumor microenvironment (TME) is often highly immunosuppressive, with factors like regulatory T cells, myeloid-derived suppressor cells, and inhibitory cytokines that dampen CAR-T cell activity. Overcoming this immunosuppression is essential for enhancing CAR-T cell efficacy. Approaches include combining CAR-T therapy with immune checkpoint inhibitors or modifying CAR-T cells to resist suppression.

⑤ CAR-T Cell Associated Toxicities

CAR-T cell therapy is associated with significant toxicities, mainly including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). CRS, characterized by excessive cytokine release, can lead to life-threatening complications. ICANS is characterized by elevated cerebrospinal fluid cytokine levels and blood-brain barrier disruption and can manifest as confusion, seizures, or even coma. Managing these toxicities requires careful monitoring and the development of strategies to mitigate their impact.

The trick of balancing efficacy and toxicity in CAR-T cell therapy is to find the “sweet spot”—potent tumor killing, minimal collateral damage. This balancing act is the heart of every clinical trial and makes antigen selection, dosing, and patient monitoring critical.

3.2 Additional Considerations – CAR-T Cell Manufacturing

Making CAR-T cells is complex. It takes time, precision, and quality control at every step.

Anti-CAR linker antibodies are used in research and development to monitor and detect CAR expression. Common linkers (like G4S and Whitlow) link the VL and VH chain of the scFv domain in the CAR. Special antibodies like anti-G4S linker mAb and anti-Whitlow linker mAb are used to detect these linkers and ensure correct CAR expression.

CUSABIO can offer anti-linker monoclonal antibodies to streamline preclinical and manufacturing QC processes.

4. Emerging Landscape and Future Strategies

4.1 Emerging Landscape of Immune Cell Therapies

The field of immune cell therapies is rapidly evolving, with several innovative approaches showing promise in treating a range of diseases, including cancer and autoimmune disorders.

Engineered immune cell therapies are produced by initially collecting blood from the patient through apheresis, isolating T cells, and employing either viral or non-viral methods to introduce a transgene that encodes a synthetic receptor. Examples of engineered T cells include:

①T cells expressing an engineered TCR consisting of TCR alpha and beta subunits

② CAR-expressing T cells (CAR-Ts) or NK cells (CAR-NKs), which consist of an extracellular antigen-binding domain fused to intracellular domains involved in TCR signaling

③ CAAR T cells (CAAR-Ts), where the chimeric receptor is comprised of an antigen-binding domain that targets autoreactive B cells

④ CAR-Tregs (Regulatory T Cells), where Tregs are isolated from peripheral blood and engineered to express a CAR that redirects them to tissue affected by autoimmune disease

4.2 Alternatives to CAR-T

4.2.1 CAR-Treg

These cells keep the immune system in check. CAR-Tregs might help prevent rejection in organ transplants or treat autoimmune diseases by calming the immune system only where needed, reducing side effects common with regular immunosuppressants.

4.2.2 CAR-NK (Natural Killer Cells)

NK cells don't need to be "matched" to the patient, so therapy could be faster and safer. They act quickly, kill broadly, and are less likely to cause graft-versus-host disease or severe side effects. They can also be produced in large numbers, making commercial therapy easier.

4.3. Future Strategies

4.3.1 Strategies to Avoid T-Cell Exhaustion

Avoiding T-cell exhaustion is crucial for sustaining CAR-T cell efficacy.

T-cell exhaustion is driven by:

① Self-aggregation or excess antigen leading to constant stimulation.

② Tonic signaling via CD28, triggering exhaustion pathways.

③ Epigenetic modifications (via EZH2) reinforces the exhausted state.

④ Expression of exhaustion-related genes such as PD-1, TIM-3, LAG3, etc.

⑤ Activation of inhibitory receptors (e.g., PD-1, TIM-3) that suppress T-cell functions.

Potential solutions:

① Use of 4-1BB co-stimulation promotes expression of memory-related genes (LEF1, TCF7, IL7R, KLF2) and oxidative metabolism, supporting long-term T-cell function.

② Suppressing or modifying CD28 signaling to reduce tonic signaling.

③ Targeting EZH2 to prevent exhaustion-promoting epigenetic modifications.

④ Blocking inhibitory receptors (e.g., PD-1/PD-L1 inhibitors).

4.3.2 Strategies to Manage Toxicities

CAR-T cells engage with tumor cells expressing target antigens, leading to the release of soluble factors that trigger pro-inflammatory responses. This cascade activates myeloid cells, which subsequently produce key inflammatory cytokines including IL-6 and IL-1β. This eventually causes inflammatory toxicities.

Current management strategies for reducing inflammatory toxicities associated with CAR-T cell therapy include:

① Corticosteroids for broad immunosuppression;

② Tocilizumab (anti-IL-6 receptor antagonist) - currently the only FDA-approved therapy for CAR-T-associated CRS;

③ Anakinra (IL-1 receptor antagonist) shows promise in combination therapy.

The prevention strategies include:

① Genetic alterations to modify CAR-T cell function

② Multiple CAR targets to improve specificity

③ Enhanced CAR design to reduce off-target effects

④ Sequestration strategies (e.g., anti-GM-CSF)

⑤ Anti-inflammatory drugs (e.g., BTKIs)

⑥ Receptor antagonists (e.g., IL-1RA)

⑦ Transcriptional inhibition (e.g., JTE-607).

Conclusion

CAR-T cell therapy stands as a beacon of hope in the fight against cancer, embodying the transformative potential of personalized medicine. Its ability to reprogram the immune system to combat malignancies has not only saved lives but also redefined the boundaries of what is possible in oncology. However, as we celebrate its successes, the journey is far from over. Challenges such as high costs, manufacturing complexities, and managing severe side effects must be addressed to ensure broader accessibility and efficacy.

As research continues to push the frontiers of this innovative therapy, CAR-T cell treatment holds the promise of a future where cancer is no longer a death sentence but a manageable condition. The story of CAR-T is not just about scientific triumph—it’s about the enduring hope for a healthier, cancer-free world.

Further Reading:

T Cell Activation - The Switch for T Cell Executive Function

How the Tumor Microenvironment Fuels Cancer's Deadly Evolution