CAR-T Cell Therapy for Solid Tumors: Key Lessons

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CAR-T Cell Therapy for Solid Tumors: Key Lessons

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CAR-T Cell Therapy for Solid Tumors: Key Lessons

Chimeric Antigen Receptor (CAR) T-cell therapy is a form of adoptive cell therapy where autologous T-cells are engineered to express CAR to specifically kill tumor cells.

Autologous CD19-targeted CAR-T cells have received approvals from major regulatory agencies including the FDA, EMA, and China’s NMPA. These CAR-T cells have transformed clinical practice, demonstrating potent anti-tumor activity.

However, developing clinically applicable CAR-T cells for solid tumor patients still faces significant hurdles, mostly owing to unknown reasons.

Several possible causes include inadequate specificity of the targeted antigens, poor trafficking, short persistence, loss of effector function, and tumor antigen heterogeneity.

Therefore, it’s crucial to draw from past CAR-T preclinical and clinical studies, analyzing the elements and challenges of CAR-T cell technology. By considering factors in basic, clinical, and practical aspects, strategies must be employed to make CAR-T technology an affordable treatment modality.

Understanding CAR-T Cell Failures in Solid Tumors

Poor Trafficking

Clinical and preclinical data have revealed two fundamental questions regarding CAR-T cell research and development: where do T cells go after intravenous injection, and how effective are they at entering tumors?

Preclinical model data indicate that few injected CAR-T cells initially enter tumors. In mouse-to-mouse T-cell transfer within same-gene mice, the T-cells only persist for a few weeks post-injection. Human trial data suggests that CAR-T cells initially accumulate in the lungs and secondary lymphoid organs, transferring very inefficiently to tumors within 24-48 hours.

The entry of CAR-T cells into tumors first requires the recognition of endothelial cell surface-secreted and binding chemokines, predominantly located in the tumor stroma rather than tumor cell-rich areas. This initial endothelial recognition is followed by selectin-mediated rolling adhesion and then firm adhesion by integrins. Driven by chemokines (especially CXCL9, CXCL10, CXCL11, and CCL5), T-cells migrate into the tumor stroma. Here, barrier cells such as perivascular cells, extracellular matrix proteins, and stromal cells (mostly fibroblasts) constitute a barrier. Some T-cells migrate through the stroma, with fewer ultimately guided by tumor-derived chemokines into tumor cell-rich areas, where they can kill tumor cells, a process that requires binding to ICAM1 on tumor cells. This process is highly inefficient, resulting in very few T-cells successfully interacting with tumor cells. There are several reasons for this inefficiency, including chemokine-CCR mismatch, adhesion receptor defects, and extracellular matrix acting as a barrier.

Another underestimated issue might be the “misdirection” of CAR-T cells toward lymphoid tissues and away from solid tumors. Manufacturing CAR T-cells have been primarily guided by data derived from testing CD19-targeted products in B-cell leukemia or lymphoma patients. These trials emphasize the importance of lymph node and/or bone marrow trafficking, expansion of anti-tumor activity, and persistence. T-cells with high levels of CCR7 and CD62L expression are known to preferentially traffic to lymph nodes or bone marrow. Therefore, most current protocols aim to generate CAR-T cells primarily displaying a central memory cell phenotype (CD62L high and CCR7 high CD45RO+), rather than an effector memory cell phenotype (CD62L low and CCR7- low CD45RO++), which is usually less conducive to migration toward tumors.

Moreover, two additional considerations should be made. Firstly, cryopreservation might affect the expression of CD62L, influencing trafficking. Secondly, CD62L is believed to promote anti-tumor activity through other mechanisms, specifically guiding T-cells into high endothelial venules within tertiary lymphoid structures present in some solid tumors. These structures might facilitate T-cell infiltration and improve anti-tumor immune responses.

Short Persistence

In clinical trials involving solid tumor patients, the persistence of CAR-T cells, as detected by PCR or flow cytometry, has shown that almost every trial conducted so far only detects CAR-T cells in blood samples. The range of CAR T-cell DNA transcripts in the blood is from 103 to 104 copies per microgram of DNA, detectable for only about a month post-infusion, with the peak appearing usually at 10-14 days. In contrast, most successful trials testing CD19-targeted CAR-T cells in leukemia patients found substantial CAR-T cells in the blood, usually between 105 and 106 copies per microgram of DNA, lasting from several months to several years.

In summary, results from preclinical and clinical studies suggest that after intravenous injection, the efficiency of CAR-T cells to transport into tumors is remarkably low, and most of this transfer likely occurs shortly after injection. Limited data from human studies suggest that the few CAR-T cells entering solid tumors have limited persistence and do not extensively proliferate.

Effector Function Loss

Consistent findings from preclinical studies of CAR-T cells reveal initially high cytotoxic activity that gradually decreases over time. Changes in the genome and epigenome are associated with this low functional state. The reasons for inducing this progressive functional decline are not entirely clear. It might be related to various factors present in the Tumor Microenvironment (TME), including low pH, hypoxia, nutritional deficiencies due to low critical amino acids and glucose levels, high levels of reactive oxygen species (ROS), the existence of immune-suppressive mediators (such as TGF-β, PGE2, adenosine, and IL-10), and interactions with bone marrow-derived suppressor cells and CD4+ regulatory T cells. Intrinsic T-cell factors include “regulatory exhaustion” mediated by immune checkpoints (such as PD-1, CTLA4, TIM3, TIGIT, and LAG3) and inhibitory intracellular signaling pathways (such as DGK, NR4A, SHP1, and cbl-b). Additionally, there are various epigenetic changes.

Tumor Antigen Heterogeneity and Antigen Spreading

Unlike B-cell malignancies or multiple myeloma, antigen expression in solid tumors is almost always lower and more heterogeneous. Therefore, the likelihood of successful treatment is low unless CAR-T cells induce bystander or antigen-spreading effects or target multiple antigens.

Lessons We Can Learned

Some solid tumor patients have found success through adoptive T-cell transfer, notably in melanoma patients with TIL therapy. The use of TIL in non-small cell lung cancer patients has also shown promising results. What can we learn from these successes to achieve greater success with CAR-T cells in solid tumors?

T-cells optimized for lymph node trafficking might not be the best cells for solid tumors

Compared to cells that are primed for peripheral sites or effector memory cells, the high expression of CD62L and CCR7 in naive or central memory T-cells can preferentially transport to secondary lymph nodes and bone marrow. When used to develop CAR-T cells targeting solid tumors, these cells with such phenotypes might be suboptimal. Solid tumor-directed CAR-T cells are designed to target full antigens expressed on solid tumor cells. Hence, when these cells enter bone marrow or lymph nodes, they will not encounter their antigens and won’t be activated to proliferate or differentiate into effector cells unless the tumor has metastasized to these locations. Data shows that effector

CAR-T cells are more effective than memory T cells in solid tumor models. Therefore, lessons indicate that standard protocols aimed at optimizing lymph node trafficking may be a key lesson to avoid for solid tumors.

DC interaction might be crucial

One critical difference between TIL or TCR-T cells and CAR T cells is that the former can be widely activated by DCs, which can provide optimal co-stimulatory signals. This co-stimulation can occur in lymph nodes and bone marrow. Finding ways to leverage DC activation to enhance CAR-T cell proliferation and sustainability is a lesson to be drawn from trials involving TIL.

Long-term persistence might not be necessary

It’s widely believed that the success of CAR-T cells is closely tied to their persistence. However, for various reasons, the importance of persistence has not been established in clinical trials testing CAR-T cells in solid tumor patients. Additionally, data from solid tumor models suggests that the persistence of CAR-T cells is associated with increased levels of T-cell dysfunction.

Ways to address this challenge include modifying CAR-T cells to make them more persistent and less prone to functional decline. Over the past few years, some encouraging successes have been achieved using this approach in solid tumor patients. However, another approach to consider is not emphasizing persistence but using a multiple dosing strategy to provide fresh CAR-T cells. Over time, the injected CAR-T cells will be depleted and can be replaced by a new dose of highly active CAR-T cells. This approach necessitates reducing the immunogenicity of CAR components to avoid rejection of the cells in repeated dosing.

Targeting multiple tumor antigens is crucial

Apart from activation mechanism differences, one crucial difference between TIL and CAR-T cells is that the former is inherently multi-clonal, and hence can target more than one antigen. Any successful CAR-T cell therapy for solid tumor patients will require targeting multiple tumor-specific antigens to optimally trigger some bystander effect or induce antigen spreading to the endogenous immune system. For instance, the design of CAR-T cells secreting FLT3 ligand recruits DCs and uses 4-1BB agonists for DC activation to enhance intrinsic T-cell response and augment anti-tumor activity.

How to Enhance CAR-T Development?

To address these challenges, it is worth considering advancing forward through two different strategies for greater success in solid tumor patients.

Strategy 1 is the current direction of the field based on the traditional “memory cell” model developed for hematological malignancy patients.

Strategy 2 employs a different “short-lived effector cell” model.

Strategy 1: Memory Cell Model

This approach aims to maximize the transport and persistence of CAR-T cells while minimizing the degree of CAR T functional decline. As part of the strategy, lymph node clearance with high-dose chemotherapy is used to enhance engraftment. The persistence and low functionality of CAR-T cells have been addressed by suppressing possible inhibitory factors by introducing specific genetic changes into T cells (such as through CRISPR), allowing T cells to “rest” to prevent chronic stimulation, or directly using armed CAR-T cells capable of secreting cytokines or other proteins to change the TME.

One major challenge associated with this strategy is the need to introduce multiple genetic changes. In recent years, many studies have been able to knock out one or more genes using standard CRISPR technology and developed more advanced CRISPR technologies that efficiently edit genes with very low off-target mutation rates.

The development of allogeneic T cells derived from induced pluripotent stem cells (iPSC) can also provide an alternative approach by allowing multiple genetic alterations to improve CAR-T cell transport and persistence. iPSCs will be initially modified using various CRISPR guides to make cells less immunogenic, then using guides for selected key inhibitors that reduce T cell function. After selecting clones with all expected changes, additional genes can be added to enhance transport or function.

Strategy 2: Short-lived Effector Cell Model

Although not well-studied, some data support the superiority of shorter-lived and more effector-like T cells in solid tumor mouse models. For example, in studies targeting mesothelin with CAR-T cells, cells with a CD28–CD3ζ intracellular domain structure (more like effectors) were found to have better anti-tumor activity than cells with a 4-1BB–CD3z intracellular domain structure.

Given that persistence is not the goal, Strategy 2 might also use mRNA-transduced CAR T cells. A notable advantage of this approach is the absence of a size limit to the transgene. It can introduce more than one mRNA through electroporation and express them simultaneously, allowing the introduction of multiple genetic variations.

However, a potential limitation of using CAR-T cells prepared using this method is the cost issue of dosing multiple CAR-T cells over time, unlike the “one-time” approach in Strategy 1. Although patients need re-infusion, it is similar to the administration of chemotherapy or immune checkpoint inhibitors, usually administered every 4-6 weeks, sometimes extending over many years.

Conclusion

Addressing the myriad challenges faced by CAR-T cell therapy in solid tumor patients is an arduous task.

Current therapies are mostly ineffective for solid tumor patients, and the reasons are not fully understood.

Obtaining better information about the transport, persistence, and function of CAR-T cells in treated patients is crucial to addressing these issues.

Successful therapies will likely require targeting a plethora of immune evasion mechanisms.

These strategies will include optimizing CAR-engineering, optimizing conditions during in vitro T-cell expansion to promote the emergence of specific T-cell subtypes, and manipulating the host to alter the TME or stimulate natural T-cells.

All these approaches might be necessary to achieve optimal outcomes.

Reference:

“CAR T cell therapy for patients with solid tumors: key lessons to learn and unlearn. Nat Rev Clin Oncol.2023 Oct 30”

(source:internet, reference only)

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