What are the types of targets for CAR-T cell therapy in solid tumors?

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What are the types of targets for CAR-T cell therapy in solid tumors?

What are the types of targets for CAR-T cell therapy in solid tumors? The antigen target of CAR T cells can be any protein, carbohydrate or glycolipid, which broadly expands the scope of target selection.

The correctly selected target plays a central role in determining the success of CAR T cell therapy.

Ideally, CAR targeting molecules should be overexpressed on tumor tissues and zero or low expression on normal cells. At the same time, the antigen target should also have plasma membrane localization.

In terms of standards, tumor-specific antigen (TSA), a tumor antigen, is the most appropriate, partly because of its unique and abundant expression on tumor cells. Since healthy tissues do not express this type of antigen, great efforts have been made to accelerate its potential application in the clinic.

However, these antigens are highly heterogeneous among patients with the same type of tumor, which in turn poses challenges for CAR T cell therapy against them. For example, due to antigen heterogeneity, determining the appropriate TSA for each patient and then being able to generate specific CAR T cells is a prerequisite. This is a very complicated and expensive process, and most patients cannot afford it.

Therefore, although TSA can be used as an ideal target for CAR T cells, successful cases of using them for CAR T therapy are rare. More effort is needed to reduce costs and simplify the procedures for accelerating their applications. Therefore, another tumor antigen called tumor-associated antigen (TAA) has been extensively explored at the same time.

However, targeting TAAs usually leads to targeted/non-tumor toxicity because they are simultaneously expressed at low levels in normal tissues, even though most TAAs are overexpressed by tumors. Fortunately, with the advancement of methods in the field of genetic engineering, this problem is gradually being resolved, leading to some TAA clinical trials for solid tumors underway. Although in most cases TSA and TAA are the first choice for solid tumor CAR T therapy, as just described, challenges often exist.

When these therapies cannot meet the application requirements, finding other types of targets becomes crucial. A class of alternative antigens including fibroblast activation protein (FAP) [28] and vascular endothelial growth factor receptor-2 (VEGFR-2) has been well studied. This type of target is not expressed on the tumor cells themselves, but is highly expressed in the tumor-associated fibroblasts of FAP or the tumor vasculature of VEGFR-2, forming a supportive niche for tumor cells. Therefore, targeting them can disrupt tumorigenesis by hindering matrix formation or angiogenesis. The literature has confirmed that it is feasible to choose FAP or VEGFR-2 as the target of CAR immunotherapy.

In addition to the above targets, there is a unique antigen, glycolipid antigen, which is also suitable as a target for CAR. This is due to the characteristics of CAR, that is, the independence of MHC. The most studied glycolipid antigen target is ganglioside GD2, which is highly overexpressed in neuroblastoma and many other types of solid tumors, and has been used as a CAR target in multiple clinical trials: NCT02992210, NCT02761915, NCT03373097 , NCT02765243).

Here, we divide the above antigens into four types of solid tumor CAR targets, namely TSA, TAA, cancer-associated stromal cell (CASC) surface antigens and glycolipid antigens, including almost all targets currently under study. As described in Table 1, we can see that the potential targets of CAR T therapy can be selected in a wide range with great flexibility.

What are the types of targets for CAR-T cell therapy in solid tumors?

The following takes representatives of the four antigen target categories as examples to introduce the selection of targets in CAR T therapy.

1. EGFRvIII is a TSA and an ideal choice for GBM-CAR T targeting

Glioblastoma (GBM) is by far the most common malignant primary brain tumor with no cure. CAR T therapy targeting GBM-related antigens (such as EGFRvIII, IL-13Rα2, HER2 and EphA2) has attracted much attention.

Epidermal growth factor receptor variant III (EGFRvIII) is the most common variant of epidermal growth factor receptor (EGFR) observed in human tumors and is caused by the in-frame deletion of part of the extracellular domain. The resulting mutant has a new sequence at the fusion junction, resulting in a tumor-specific and immunogenic epitope that is not expressed in normal tissues.

This mutant is not only expressed in most GBM patients, but also in other malignant tumors. It is worth noting that EGFRvIII is found to be very common in CD133+ glioblastoma cancer stem cells and endows EGFRvIII+/CD133+ cells with a high degree of self-renewal and tumor initiation ability. At the same time, EGFRvIII as a constitutively active tyrosine kinase plays an important role in tumorigenesis and invasion.

These features, including surface neoantigens specifically expressed on malignant cells, high expression in GBM and cancer stem cells, and the ability to induce phenotypic transformation into malignant tumors, make EGFRvIII an ideal target for CAR-modified T cells to treat GBM. point.

Based on the above-mentioned properties of EGFRvIII, a second-generation CAR derived from murine 3C10 single-chain variable fragment (scFv) fused with 4-1BB and CD3 was produced and performed in subcutaneous and orthotopic xenograft models of human EGFRvIII-positive GBM test.

The results show that specific targeting of EGFRvIII and lack of reactivity to wild-type EGFR can significantly delay tumor progression. According to reports, a clinical trial of the University of Pennsylvania using anti-EGFRvIII CAR T cells to treat 10 EGFRvIII-positive GBM patients is safe, and there is no evidence of extra-tumor toxicity, CRS, and cross-reactivity to wild-type EGFR. Other forms of EGFRvIII-oriented CAR have also shown satisfactory results in preclinical models and the first human exploratory study of newly diagnosed GBM patients. However, it is also important to note that although the above data demonstrates the successful transport of CAR T cells to the tumor and effective antigen targeting, evaluation of tumor tissue resected at relapse shows that EGFRvIII expression is lost over time.

The characteristics of antigen loss and intratumoral and inter-individual heterogeneity in GBM increase the difficulty of CAR T cell therapy. In this case, alternative targets should be found to reduce the chance of immune escape and enhance the anti-tumor immune response

2. Mesothelin is a TAA, suitable for CAR T targeting of a variety of solid tumors

Tumor-associated antigens (TAA) are potential candidates for immunotherapy targets because they are overexpressed on tumor cells, but are almost not expressed in most normal tissues. As a kind of TAA, mesothelin is highly expressed in a wide range of solid tumors, such as epithelioid mesothelioma, extrahepatic cholangiocarcinoma, pancreatic ductal adenocarcinoma (PDAC), ovarian cancer and gastric cancer.

In addition, the reported data indicate that increased expression is associated with poorer prognosis in patients with ovarian cancer, cholangiocarcinoma, pancreatic cancer, triple-negative breast cancer, and lung adenocarcinoma. Regarding its expression in normal tissues, it is limited to the mesothelial cell layer, while its expression in the epithelial cell layer is less.

Mesothelin is a cell surface glycoprotein with unclear physiological functions, but it may be a key factor in malignant tumors, because its abnormal expression plays an important role in malignant transformation and tumor invasion by promoting cancer cell proliferation, metastasis and invasion . The above-mentioned characteristics of mesothelin make it an attractive candidate for therapeutic target.

In the past few decades, strategies for mesothelin have been developed in preclinical research and early clinical trials, including immunotoxin-carrying proteins, the use of monoclonal antibodies, antibody-drug conjugates, specific vaccines and CARs T cells. Among these strategies, CAR T cell therapy has attracted much attention due to its huge potential applications in the clinic, and a number of preclinical studies are underway.

Jiang et al. showed that anti-mesothelin CAR T cells can inhibit tumor growth and penetrate a xenograft model derived from patients with mesothelin-positive PDAC. Engineered CAR T cells expressing TCR with enhanced affinity for mesothelin were also evaluated in PDAC and demonstrated effective tumor site infiltration and tumor cell death and prolonged survival of treated mice.

Aiming at the anti-tumor effect of mesothelin-specific CAR T cells, researchers have carried out clinical trials in patients with epithelial ovarian cancer, malignant epithelial mesothelioma and PDAC, and achieved direct anti-tumor effects in clinical trials, NCT02159716 (clinicaltrials.gov). In another phase I trial, the activity of mesothelin-specific CAR T cells on pancreatic cancer metastasis was evaluated, and the results showed the safety, feasibility and therapeutic potential of CAR to recognize mesothelin.

It is worth noting that various mesothelin-specific CARs are being studied in ongoing trials, such as NCT02414269 and NCT02465983 (clinicaltrials.gov). All these results indicate that mesothelin is an effective target for CAR T treatment in solid tumors expressing mesothelin.

3. Ganglioside GD2, a surface glycolipid antigen CAR target

Ganglioside GD2-specific CAR contrasts with the protein antigen of engineered CAR T cells because GD2 is a glycolipid found on the outer cell membrane. GD2 is highly overexpressed in a variety of tumor cells, including neuroblastoma, astrocytoma, retinoblastoma, Ewing’s sarcoma, rhabdomyosarcoma, small cell lung cancer, melanoma, and breast cancer, but in normal tissues (including The expression is limited in the central nervous system, mainly in neuronal cell bodies and mesenchymal stem cells, and the expression level is low on peripheral nerves and skin melanocytes.

Reported data show that its expression density on the cell membrane of neuroblastoma can reach 5-10 million molecules/cell. In addition, the level of circulating GD2 is not sufficient to interfere with the binding of its specific monoclonal antibody in the circulation. These characteristics make it an ideal target for CAR T cells.

So far, anti-GD2-CAR has been fully studied in preclinical plans and clinical trials for various diseases such as neuroblastoma, osteosarcoma and melanoma. In a study to test the cytotoxicity of anti-GD2 CAR T cells in melanoma, the results revealed the specific lysis of GD2 positive melanoma cells in vitro. In two patient-derived xenograft (PDX) models, rapid tumor regression was observed in mice that received intravenous or local intratumoral injection of anti-GD2 CAR T cells.

Therefore, the researchers concluded that anti-GD2CAR T cells can not only effectively lyse melanoma in a GD2-specific manner, but also release Th1 cytokines in vitro and in vivo, which represents a potential strategy for the future treatment of melanoma patients.

Recently, the potential anti-tumor efficacy of anti-GD2 CAR T cells in H3-K27M+ diffuse midline glioma (DMG) has also been reported. In this study, anti-GD2 CAR T cells showed strong antigen-dependent cytokine release and DMG cell killing effect in vitro. In the five PDX models, systemic administration of GD2-CAR T cells cleared the transplanted tumor.

Based on the accumulated data, a number of clinical trials such as NCT02992210, NCT02761915, NCT03373097 and NCT02765243 (clinicaltrials.gov) are currently using GD2 targeting CAR in various solid tumors.

4. FAP, CAR target on the surface of cancer-related fibroblasts

Most CAR T cells are genetically modified to target antigens on cancer cells. However, some antigen targets expressed on the surface of non-malignant cancer-associated stromal cells (CASC) are also suitable for CAR T cells. An attractive candidate for these targets is FAP, a transmembrane serine protease, which is highly expressed on CASC in more than 90% of epithelial cancers and is low expressed in healthy adult tissues.

There are several advantages to choosing this type of target. First, stromal cells are genetically more stable than cancer cells. Therefore, it is easier to target stromal cells in a stable manner using the designated antigen target. Secondly, tumor stroma has the function of supporting tumor cell growth, invasion and angiogenesis to form a physical barrier against targeted tumor immunotherapy, and build an immunosuppressive niche by attracting immunosuppressive cells, regulating T cell function and expressing inhibitory molecules .

Targeting stromal cells impairs these functions and at the same time hinders tumor growth. Third, the mechanism by which tumor stroma supports tumor growth is common. Therefore, targeted therapy for this mechanism may be possible for a wide range of tumors.

So far, several groups have reported their results using anti-FAP CAR T cells. A study by Wang et al. developed an anti-mouse FAP-CAR construct containing scFv derived from mAb 73.3 with a framework of CD8α-4-1BB-CD3ζ. In vitro, the transduced FAP-CAR T cells secrete interferon-γ and specifically kill 3T3 cells expressing FAP. In mice adoptively transferred with 73.3-FAP-CART cells, FAPhi stromal cells were reduced, and the growth of subcutaneously transplanted tumors was inhibited.

In addition, the results showed that after FAP-CAR T treatment, the extra-tumor toxicity in their model was minimal. Therefore, the researchers concluded that treatment with 73.3-FAP-CAR T cells targeting FAP-expressed tumor stroma is safe and effective, and recommended further clinical development of anti-human FAP-CAR. Based on the data obtained in this study, the researchers conducted a follow-up investigation on the effect of FAP-CAR T cells on tumor-induced connective tissue hyperplasia.

In a tumor model that promotes connective tissue proliferation, FAP-CAR T cells reduce tumor growth, and are accompanied by the destruction of connective tissue matrix, angiogenesis and cancer cell proliferation. Despite the encouraging data observed, the work of Tran et al.

It revealed serious side effects caused by the administration of FAP-CAR T cells produced by FAP-specific mAbs FAP5 and sibrotuzumab. The results not only show a limited effect on tumor growth in a wide range of murine models, but also cause morbidity and mortality in most mice.

The difference between different groups may be related to the specificity and affinity of the scFv used. Regardless of these comparison results, FAP targeting CAR T therapy has entered the Phase I clinical trial NCT01722149 for patients with malignant pleural mesothelioma sponsored by the University of Zurich (clinicaltrials.gov).

A carefully selected antigen target not only helps to reduce side effects and partially overcome the TME barrier of solid tumors, but also plays a decisive role in the ultimate anti-tumor efficacy. The immunosuppressive components in TME are also good candidate targets, because under appropriate treatment conditions, their hostile functions can be subverted, which is conducive to tumor eradication.

When designing a CAR structure with expected effectiveness, the selected target and TME are interrelated factors that need to be carefully considered. The type of tumor determines its specific TME, so specific goals are required. Elucidating the mechanism of interaction between the target and TME will help develop new therapies using CAR-modified T cells.

(source:internet, reference only)

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