October 15, 2021

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Research progress of CAR-T treatment of Ewing’s sarcoma (EWS)

Research progress of CAR-T treatment of Ewing's sarcoma (EWS)

Research progress of CAR-T treatment of Ewing’s sarcoma (EWS)



Research progress of CAR-T treatment of Ewing’s sarcoma (EWS).

Ewing sarcoma (EWS) is a malignant tumor of bone or soft tissue, which mainly occurs in children and young people.

It is the second most common primary bone tumor after osteosarcoma.

Traditional treatments, including active neoadjuvant therapy and adjuvant chemotherapy combined with surgery and/or radiotherapy, have greatly improved the long-term survival rate of patients, with the 5-year survival rate exceeding 70%.

However, once the tumor cells metastasize or recur, patients often show a poor prognosis.

In the past ten years, new immunotherapies have emerged one after another, such as immune checkpoint blockers and therapeutic cancer vaccines. Among them, CAR-T therapy is a revolutionary treatment method. It expresses and synthesizes chimeric antigen receptor (CAR) on T cells through genetic engineering. It has become a “routine” method for cancer treatment worldwide.

Recently, CAR-T therapy has also been used for solid tumors, including its application in primary bone tumors. CAR-T therapy may play a superior anti-tumor effect in these EWS patients.


CAR-T cell

CARs are composed of four components: extracellular antigen recognition domains, the most common being single-chain variable fragments (scFv); structural components, such as hinges and transmembrane domains; and provide common functions for maintaining the effector functions of CAR-T cells. Stimulation signal domain, and CD3ζ activation domain.

Research progress of CAR-T treatment of Ewing's sarcoma (EWS)

The first-generation CARs usually only contain the CD3-ζ chain signal transduction domain. The addition of a costimulatory molecule to the first-generation CARs produces so-called second-generation CARs, while the third-generation CARs include the addition of two costimulatory molecules to the first-generation CARs.

This method not only enables the immune cells to be targeted, but also overcomes the immune tolerance dilemma, so that the modified immune cells have strong anti-tumor activity.

CAR-T cell-based therapies show strong anti-tumor activity without being restricted by the traditional major histocompatibility complex (MHC).

After transplantation, CAR-T cells can proliferate in large quantities and exhibit long-lasting anti-tumor activity.


CAR-T cells targeting EWS antigen

The effective application of CAR-T cells in tumor therapy requires specificity for tumor cells. Therefore, finding tumor-specific antigens is a very important task.

Currently, the targets identified in EWS include VEGFR2, IGF1R, ROR1, GD2, B7-H3 (CD276), EphA2 and NKG2D ligands. These receptors can be used as effective therapeutic targets for CAR-T cells to treat EWS and inhibit metastasis.

Research progress of CAR-T treatment of Ewing's sarcoma (EWS)

VEGFR2

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that can induce physiological and pathological angiogenesis.

VEGF receptors (VEGFRs) include three types: VEGFR1, VEGFR2 and VEGFR3, among which VEGFR2 plays a major role in regulating endothelial cell VEGF signal transduction.

VEGF-mediated signaling pathways have been shown to exist in tumor cells and play a key role in tumorigenesis.

In a preclinical study, VEGFR2-specific CAR-T cells can effectively lyse VEGFR2-positive cells and produce effective antigen-specific degranulation, cytokine secretion, and proliferation responses. Therefore, VEGFR2 may be a suitable target for CAR-T cell therapy in EWS.

CAR-T cells targeted by VEGFR2 have been used in early clinical trials for metastatic melanoma and epithelial cancer (NCT01218867), but the current results show poor clinical response. Therefore, further research on the effect of VEGF signaling pathway on EWS treatment is very important.

IGF1R

IGF1R is a tetrameric transmembrane receptor tyrosine kinase. The binding of the ligand to the IGF1Rα subunit leads to the autophosphorylation of the β subunit and the recruitment of adaptor proteins, which ultimately leads to the activation of the signal cascade, which in turn promotes proliferation, survival, transformation, metastasis and angiogenesis.

Since IGF1R is expressed in EWS, many experiments use it as an immune target for EWS treatment.

An in vitro study showed that CAR-T cells targeted by IGF1R showed specific cytotoxicity, mainly releasing IFN-γ, TNF-α and IL-13 for sarcoma.

In addition, CAR-T cells targeted by IGF1R are also conducive to the prolonged survival of local sarcoma models. Although CAR-T cells targeted by IGF1R have certain anti-tumor activity, IGF1R is also expressed in normal tissues. It is not clear whether they have any toxic side effects on the body.

In a phase II study of EWS, researchers found that patients experienced adverse events such as neutropenia and leukopenia after treatment with ganitumab (a fully human anti-IGF1R antibody). According to reports, lymphocytes and monocytes have low cell surface IGF1R expression, which makes them difficult to be recognized by CAR-T cells targeted by IGF1R.

In addition, the off-target toxicity can be solved by changing the affinity of CAR-T cells to target cells or adjusting the therapeutic dose of CAR-T cells. However, before realizing its clinical application, the non-targeted toxicity of IGF1RCAR-T cells should be systematically evaluated.

ROR 1

ROR1 is one of twenty different RTK families and consists of three different extracellular domains, including immunoglobulin-like domains, cysteine-rich (CRD) and Kringle (KNG) domains, and intracellular TK domain.

ROR1 is not expressed in normal human tissues, but is overexpressed in several human malignant tumors, and may be used as a survival factor for tumor cells.

The anti-sarcoma activity of ROR1 targeting CAR-T cells in vitro and in vivo has been confirmed. In addition, the safety of ROR1 targeting CAR-T cells has been verified in primates. However, recent studies have shown that the expression of ROR1 is not specific to tumor tissues.

Cell surface ROR1 has been observed in several areas of human parathyroid glands, pancreatic islets, and intestinal tract, and it is especially abundant in gastric antrum and gastric corpus.

Shivani Srivastava et al. designed a logic-gated ROR1CAR-T that can solve the problem of off-target toxicity. In this design, CAR-T cell activity only eliminates tumors that express antigens A and B at the same time.

The synthetic Notch receptor for antigen A can induce the expression of CAR for specific antigen B, and regulate the function of T cells by integrating multiple signals, so that tumor tissues and normal tissues can be more accurately distinguished.

Although the above studies have proved that CAR-T cells targeted by ROR1 have a certain efficacy in the treatment of EWS, CAR-T cells targeted by ROR1 have not yet entered clinical trials, and the latest progress is under recruitment (NCT02706392), which shows great Development potential.

GD2

GD2 is a cell surface molecule with a strictly restricted expression pattern and is highly expressed in EWS. Due to the limited distribution of GD2 in normal tissues, immune targeting is safe.

One study proved that GD2-targeted CAR-T cells have a strong cytolytic response to EWS cells, and another study also confirmed the anti-tumor activity of GD2-targeted CAR-T cells on EWS.

However, other studies have shown that CAR-T cells targeted by GD2 alone cannot eliminate metastatic or in situ injected EWS cells.

In a phase I study, researchers used GD2 targeting CAR-T cells to treat patients with neuroblastoma and found that GD2 targeting CAR-T cell therapy alone had no objective clinical response.

Therefore, enhancing the anti-tumor effect of GD2 targeting CAR-T cells is the key to its successful clinical application.

One strategy is to combine GD2 with other feasible targets to construct T cells expressing multiple CARs. Another method can combine GD2-targeted CAR-T cells with immune checkpoint inhibitors to improve efficacy.

B7-H3 (CD276)

B7-H3 is a member of the B7 immunomodulatory protein family and is overexpressed in a variety of solid tumor cells. B7 protein binds to CD28/CTLA-4 family members and acts as a costimulatory signal in T cell activation.

In addition, B7-H3 is overexpressed during pathological angiogenesis, which may make it an attractive target for selective destruction of tumor vasculature.

In recent years, the use of B7-H3 to target CAR-T cells to treat solid tumors has received extensive attention. In the in vitro orthotopic and metastatic xenografts of pancreatic ductal adenocarcinoma, ovarian cancer and neuroblastoma mouse models, B7-H3 targeting CAR-T cells showed good curative effect without obvious adverse reactions.

Robbie GM et al. tried to use B7-H3 targeted CAR-T cells to treat solid tumors in children. They found that more than 90% of children’s sarcomas tested had high expression of B7-H3 and EWS.

Further experiments have shown that CAR-T cells targeted by B7-H3 can eliminate EWS xenografts in vivo, and have a significant survival advantage compared with mouse treatment controls. Therefore, B7-H3 targeting CAR-T cells may be a viable option for the treatment of EWS.

In addition, the combination of B7-H3 targeting CAR-T cells with immune checkpoint inhibitors or traditional chemotherapy drugs seems to be a viable option.

EphA2

Members of the Eph family are involved in cell transformation, metastasis and angiogenesis. There are two types of Eph receptor ligands: ephrin-A and ephrin-B. EphA2 is overexpressed in a variety of cancers, including breast cancer, melanoma, and prostate cancer.

Related studies have shown that EphA2 is up-regulated in EWS cells and participates in the migration of endothelial cells to tumors to assist tumor angiogenesis.

Studies have shown that CAR-T cells targeted by EphA2 can effectively kill EWS cells in mice and are related to prolonging survival time. However, only a small part of EWS cells express EphA2, and the anti-tumor activity of EphA2CAR-T cells in mice may affect the growth and metastasis of EWS mainly by acting on tumor angiogenesis.

NKG2D

NKG2D is a powerful activation receptor expressed by natural killer (NK) cells and T cells. In recent years, the role of NKG2D and its ligands in EWS has attracted more and more attention.

Manfred Lehner et al. constructed NKG2D-specific CAR-T cells through lentiviral transduction or mRNA transfection. These CAR-T cells can effectively eliminate EWS cells in vitro. In addition, histone deacetylase inhibitors can up-regulate the expression of NKG2D ligand in EWS. Therefore, the combination of NKG2D-specific CAR-T cells and histone deacetylase inhibitors is considered an effective treatment for EWS.

Research progress of CAR-T treatment of Ewing's sarcoma (EWS)


Summary and outlook

CAR-T cell therapy is a promising immunotherapy method, and its therapeutic effect on blood and lymphatic system tumors is encouraging. At the same time, people are also full of expectations for CAR-T cell treatment of solid tumors. EWS is an aggressive childhood cancer. CAR-T therapy may play a superior anti-tumor effect in EWS patients. Some CAR-T cell therapy EWS research has made some encouraging progress.

At present, the epitopes used for CAR-T cells mainly include VEGFR2, IGF1R, ROR1, GD2, B7-H3, EphA2 and NKG2D. Although CAR-T cells targeting these targets exhibit anti-tumor activity in in vitro or in vivo preclinical models, these targets also have their limitations, such as off-target toxicity, insufficient action, and low expression. Most studies are still in progress. In the early stage, it is far from clinical application.

To solve the above problems, CAR-T cell therapy should pay attention to the following aspects: first, to find more specific targets for tumors; secondly, when the target is highly expressed in tumor cells and low in normal cells, control The treatment threshold can reduce damage to normal tissues. Third, the affinity between CARs and homologous antigens can be adjusted to achieve targeting.

The obstacles to CAR-T cell therapy, such as tumor penetration, anti-killing, antigen escape, and immunosuppression, can be resolved from the following aspects.

1) CAR-T cells can be designed to express chemokine receptors to recognize the up-regulated chemokines in TME, thereby increasing the infiltration of CAR-T cells.

2) The combination of CAR-T cells with conventional therapies or immune checkpoint inhibitors may be worth exploring.

3) Constructing immune cells expressing multiple CARs or combining multiple CAR-T cells can provide higher tumor cell destruction efficiency.

4) More co-stimulatory expression receptors can be introduced into CAR-T cells, and CAR-T cells that target tumor antigens and immunosuppressive cytokines or immunosuppressive cells in TME can be constructed to resist T cell tumors Immunosuppressive effect.

5) Combining certain drugs can up-regulate the expression of antigens in tumors through epigenetics, and enhance the killing effect of CAR-T cells on tumors.

References: Research progress of CAR-T treatment of Ewing’s sarcoma (EWS)

1.A Novel Treatment for Ewing’s Sarcoma: Chimeric Antigen Receptor-T Cell Therapy. Front Immunol. 2021;12: 707211.

Research progress of CAR-T treatment of Ewing’s sarcoma (EWS)

(source:internet, reference only)


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